1. 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

2. 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

3. Separating organelles
Differential centrifugation
Density gradient centrifugation

4. Density Gradient Centrifugation
Normal Centrifugation

5. 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,

6. 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.

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

8. 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

9. Cell culture environment
Basal Media
Maintain pH and osmolarity ( 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

10. 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

11. 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

12. 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

13. 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

14. 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.

15. 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

16. 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)

17. 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%

18. 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

19. 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)

20. 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

21. 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

22. 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

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

24. 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.

25. 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.

26. 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.

27. Methodology for Cell Culture

28. 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

29. 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

30. 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

31. 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

32. Cell Morphologies Vary Depending on Cell Type
Fibroblastic
Epithelial
Endothelial
Neuronal

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

34. 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.
FW Went demonstrated that there were growth substances in coleoptiles from oats.

35. 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?).

36. History Leads Cell Culture
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.
Successful continuously growing cultures of carrot and tobacco.
Formation of adventitious shoots and roots in tobacco.
Miller and Skoog discovered kinetin, a cytokinin that plays an active role in organogenesis.
1954 – First plant grown from a single cell.

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

38. 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).

39. 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.

40. 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.

41. 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

42. 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

43. 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

44. Lab to Land

45. 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.

46. 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”

47. 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 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