SoS, Dept. of Biology, Lautoka Campus Topic 2: Cell Line Theory BIO508 Cell Biology Slide Design: Copyright © McGraw-Hill Global Education Holdings, LLC. Lecturer: Dr.Ramesh Subramani Topic 2: Cell Line Theory

Purification of Cell Organelles Cell disruption Separation of different organelles using centrifugation Breaking down of Plasma Membranes In Cells: Cells are suspended in isotonic sucrose Sonication Homogenization Cells in hypotonic solution – rupture of cell membranes

Separating organelles Differential centrifugation Density gradient centrifugation

Density Gradient Centrifugation Normal Centrifugation

Cell Culture Requirements Solid Media Specially coated plastic dishes or flasks (CAMs’) Agar as the medium Growth Media Rich in nutrients- amino acids, vitamins, salts fatty acids, glucose, serum provides the different growth factors,

Requirement for Cell Growth Appropriate tissue (some tissues culture better than others) A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant tissue and can over run a culture Growth regulators, both auxins & cytokinins.

CO2 incubator Maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

Cell culture environment Substrate or liquid (cell culture flask or scaffold material) Chemically modified plastic or coated with ECM proteins suspension culture Environment (CO2, temperature 37oC, humidity) Oxygen tension maintained at atmospheric but can be varied Sterility (aseptic technique, antibiotics and antimycotics) Mycoplasma tested

Cell culture environment Basal Media Maintain pH and osmolarity (260-320 mOsm/L). Provide nutrients and energy source. Components of Basal Media Inorganic Salts Maintain osmolarity Regulate membrane potential (Na+, K+, Ca2+) Ions for cell attachment and enzyme cofactors pH Indicator – Phenol Red Optimum cell growth approx. pH 7.4 Buffers (Bicarbonate and HEPES) Bicarbonate buffered media requires CO2 atmosphere HEPES Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2) Glucose Energy Source

Cell culture environment Components of Basal Media Keto acids (oxalacetate and pyruvate) Intermediate in Glycolysis/Krebs cycle Keto acids added to the media as additional energy source Maintain maximum cell metabolism Carbohydrates Energy source Glucose and galactose Low (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media Vitamins Precursors for numerous co-factors B group vitamins necessary for cell growth and proliferation Common vitamins found in basal media is riboflavin, thiamine and biotin Trace Elements Zinc, copper, selenium and tricarboxylic acid intermediates

Cell culture environment Supplements L-glutamine Essential amino acid (not synthesised by the cell) Energy source (citric acid cycle), used in protein synthesis Unstable in liquid media - added as a supplement Non-essential amino acids (NEAA) Usually added to basic media compositions Energy source, used in protein synthesis May reduce metabolic burden on cells Growth Factors and Hormones (e.g.: insulin) Stimulate glucose transport and utilisation Uptake of amino acids Maintenance of differentiation Antibiotics and Antimycotics Penicillin, streptomycin, gentamicin, amphotericin B Reduce the risk of bacterial and fungal contamination Cells can become antibiotic resistant – changing phenotype Preferably avoided in long term culture

Cell culture environment Foetal Calf/Bovine Serum (FCS & FBS) Growth factors and hormones Aids cell attachment Binds and neutralise toxins Long history of use Infectious agents (prions) Variable composition Expensive Regulatory issues (to minimise risk) Heat Inactivation (56oC for 30 mins) – why? Destruction of complement and immunoglobulins Destruction of some viruses (also gamma irradiated serum) Higher heat can damage growth factors, hormones & vitamins and affect cell growth

Hemacytometer Specialized chamber with etched grid used to count the number of cells in a sample. use of trypan blue allows differentiation between living and dead cells

Using the Hemacytometer Remove the hemacytometer and coverslip (carefully) from EtOH and dry thoroughly with a kimwipe. Center coverslip on hemacytometer Barely fill the grid under the coverslip via the divet with your cell suspension. Count cells in ten squares (5 on each side) by following diagram at station.

How the cells will appear Bright refractile “spheres” are living cells, Blue cells about the same size as the other cells are dead. Keep a differential count of blue vs. clear for viability determination. Sometimes there will be serum debris, and this will look red or blue and stringy or gloppy--don’t count it! Blood cells

Calculate your cells/ml Calculate the number of total cells in one ml of your suspension. Total cells counted x (dilution factor) x (10,000) number of squares Here, dilution factor is 2 and # of squares is 10 (our example 62/10 x2 x104 =1.24 x 105)

Determine your percent viability Viability is a measure how many of your cells survived your cell culture technique. # of viable (living) cells x 100 total number of cells counted Our example 54/62 x 100 =87.09%

Culturing cells in the laboratory Revive frozen cell population Isolate from tissue Containment level 2 cell culture laboratory Maintain in culture (aseptic technique) Typical cell culture flask Sub-culture Count cells Used to freeze cells Cryopreservation

Cryopreservation of Cell Lines The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times Reduced risk of microbial contamination Reduced risk of cross contamination with other cell lines Reduced risk of genetic drift and morphological changes Work conducted using cells at a consistent passage number Reduced costs (consumables and staff time)

Cell culture Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells. Cell culture was first successfully undertaken by Ross Harrison in 1907. Roux in 1885 for the first time maintained embryonic chick cells in a cell culture

Significance of cell culture Areas where cell culture technology is currently playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells

Significance of cell culture Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene

Tissue culture In vitro culture (maintain and/or proliferate) of cells, tissues or organs Types of tissue culture Organ culture Tissue culture Cell culture

Organ culture The entire embryos or organs are excised from the body and culture Advantages Normal physiological functions are maintained. Cells remain fully differentiated. Disadvantages Scale-up is not recommended. Growth is slow. Fresh explantation is required for every experiment.

Tissue Culture Fragments of excised tissue are grown in culture media Advantages Some normal functions may be maintained. Better than organ culture for scale-up but not ideal. Disadvantages Original organization of tissue is lost.

Cell Culture Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture. Advantages Development of a cell line over several generations Scale-up is possible Disadvantages Cells may lose some differentiated characteristics.

Methodology for Cell Culture

Types of Cell Culture Primary cultures: Derived directly from animal tissue - embryo or adult? Normal or neoplastic? Cultured either as tissue explants or single cells Initially heterogeneous – become overpopulated with fibroblasts Finite life span in vitro Retain differentiated phenotype Mainly anchorage dependant Exhibit contact inhibition

Types of Cell Culture Secondary cultures: Derived from a primary cell culture Isolated by selection or cloning Becoming a more homogeneous cell population Finite life span in vitro Retain differentiated phenotype Mainly anchorage dependant Exhibit contact inhibition

Types of cell culture Continuous cultures: Derived from a primary or secondary culture Immortalised: Spontaneously (e.g.: spontaneous genetic mutation) By transformation vectors (e.g.: viruses & plasmids) Serially propagated in culture showing an increased growth rate Homogeneous cell population Loss of anchorage dependency and contact inhibition Infinite life span in vitro Differentiated phenotype: Retained to some degree in cancer derived cell lines Very little retained with transformed cell lines Genetically unstable

Isolation of cell lines Resected Tissue Cell or tissue culture in vitro Primary culture Secondary culture Sub-culture Cell Line Immortalization Successive sub-culture Single cell isolation Clonal cell line Senescence Transformed cell line Immortalised cell line Loss of control of cell growth

Cell Morphologies Vary Depending on Cell Type Fibroblastic Epithelial Endothelial Neuronal

A comparison Phase contrast microscopy Light microscopy Can be used on living cells requires stain, thus killing cells

History Leads Cell Culture 1838 – Schwann and Schleiden put forward the theory which states that cells are totipotent, and in principle, are capable of regenerating into a complete plant. Their theory was the foundation of plant cell and tissue culture. 1902 – Haberlandt conducted the first but unsuccessful attempt of tissue culture using monocotyledons. 1926 - FW Went demonstrated that there were growth substances in coleoptiles from oats.

Went’s Experiment on plant tissue culture A. Germination of an oat seed B. Decapitate tip of coleoptile and place on agar block. C. Agar block is placed on top of the decapitated tip of the seedling (what happens?). D. Agar block is placed on the side of the leaf on the decapitated tip of the seedling (what happens?).

History Leads Cell Culture 1934 - White generated continuously growing culture of meristematic cells of tomato on medium containing salts, yeast extract, sucrose and 3 vitamins (pyridoxine, thiamine, nicotinic acid). This established the importance of additives in tissue culture. 1939 - Successful continuously growing cultures of carrot and tobacco. 1948 - Formation of adventitious shoots and roots in tobacco. 1953 - Miller and Skoog discovered kinetin, a cytokinin that plays an active role in organogenesis. 1954 – First plant grown from a single cell.

History Leads Cell Culture 1957 - Discovery that root or shoot formation in culture depends on auxin:cytokinin ratio. 1962 - Development of the Murashige and Skoog (MS) medium. In the 70’s and 80’s - beginning of genetic engineering. Folke K. Skoog 1908–2001

Tissue Culture Tissue culture is the term used for “the process of growing cells artificially in the laboratory” Tissue culture involves both plant and animal cells. Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture).

Plant Tissue Culture- Step I Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue. This tissue must be sterilized to remove microbial contaminants.

Plant Tissue Culture- Step II Establishment of the explant in a culture medium. The medium sustains the plant cells and encourages cell division. It can be solid or liquid. Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error.

Plant Tissue Culture- Step III Multiplication- the explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin (high) ratios to form multiple shoots The callus may be subdivided a number of times Dividing shoots Warmth and good light are essential

Plant Tissue Culture- Step IV Root formation - The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios The bottles on these racks are young banana plants and are growing roots

Plant Tissue Culture- Step V The rooted shoots are potted up (de-flasked) and ‘hardened off’ by gradually decreasing the humidity This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss Tissue culture plants sold to a nursery & then potted up

Lab to Land

Significance of Plant Tissue Culture A single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars. Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely. Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year. In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants. Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture.

Significance of Plant Tissue Culture Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner). Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then cloned. Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability. “Commercial, Research and Breeding Purposes”

Acknowledgements… Any Questions?? The teaching material used in this lecture is taken from: JB Reece, LA Urry, ML Cain, SA Wasserman, PV Minorsky and RB Jackson. 2011. Campbell Biology (9th Edition), Publisher Pearson is gratefully acknowledged. Some information presented in this power point lecture presentation is collected from various sources including Google, Wikipedia, research articles and some book chapters from various biology books. Material and figures used in this presentation are gratefully acknowledged. This material is collected and presented only for teaching purpose. Any Questions?? Dr.Ramesh Subramani, Assistant Professor in Biology