What we learned so far…

• Basic differences between plant and animal cell culture

• What are different requirements of animal cell culture

(Equipment, media & sera, cell culture source, ethical approvals)

• Basic terminology
(Primary culture, explant culture, suspension culture, adherent cells (anchorage dependent cells), contact
inhibition, crisis, senescence, transformed cells, immortal cells, established cell lines, fibroblast cells,
epithelial cells, spontaneous immortalization, sub culturing cells, passage number, spilt ratio, trypsinization,
seeding density)

• Little bit about what we can do with animal cell cultures

(importance and applications of culture in research: homogeneous population of cell for experimentation,
reduction in the number of animal killing)

• Characterizations of cell cultures

(using HNGC cell line example: doubling time, explant culture, primary culture, Hayflick limit, cellular
markers, spontaneous immortalisation, chromosomal abnormalities etc.

• Cell counting and viability estimation in response to drug

 In this Lecture…

• Cell culture media and components….

• Culture phase and routine maintenance…
• Contamination in cell cultures.. types, detection and mitigation
• Cryopreservation of cells
• Transformation and immortalization of cells
• Application of cell culture : Drug screening and toxicity testing
 Media type
Defined : Serum free or special media
Semi defined : Serum supplemented media

Most common media

Eagle’s MEM: Minimal Essential Medium

DMEM: Dulbecco’s Modified Eagle’s Medium

RPMI 1640: Roswell Park Memorial Institute

Most common sera

FBS: Foetal Bovine Serum
 Media components
Buffer components : NaHCO3, CO2, HEPES

pH indicator: phenol red

Glucose : energy source (glycolysis)

Amino acids : essential, Glutamine

Vitamins : ‘B’ group, folic acid, biotin (water soluble)

Salts and trace elements : Na+, K+, Mg2+, Ca2+, Cl-, SO42-, PO43-, HCO3-,
Se, Pyruvate etc.
DNA bases & nucleosides:

Proteins and peptides: Insulin, transferrin, albumin

Lipids: Steroids

Serum : provides hormones and growth factors, trypsin

inhibition, peptides, lipid and cholesterols
Basal components
Eg DMEM
 Culture phase

Stationary Phase
106
Cell density (cells/ mL)

Decline Phase

Growth (log) Phase

105

Lag Phase
0 5 10
Time (days)
 Growth phases

• Lag phase
• Log phase
– Cells still dividing and spreading out
• Plateau phase
– Limited space to grow
– Limited nutrients
– Toxicity build-up
Which image is 70-80% confluent?
1. Left
2. Middle
3. Right
4. None
 Epithelial Cells in Culture

Move and Grow in patches

1 2 3
 Fibroblast Cells in Culture

Tend to grow and move solitary Do not want to form patches

1 2 3
 Primary Culture
Isolation of tissue (Biopsy, animal tissue/ organ)

Dissection (selection of required tissue; removal of fatty and necrotic tissue)

Fine Dissection Mechanical disaggregation Enzymatic dissociation

Primary Explant Dispersed primary culture single cell suspension

Cellular outgrowth

Subculture

Cell line
 Explant

Culture of animal cells, R. I. Freshney

Routine Maintenance

70-80 % Confluent
 Maintenance
• Media changes
– Reduce by-products and toxin build up
– Removal of detached (dead cells)
– Nutrient depletion
• Cells washed
– Phosphate buffered saline solution
– Hanks buffered salts solution
Subculture (adherent cells)
Fate of primary culture
HAYFLICK LIMIT
Telomeres and Replicative
Senescence
TTAGGG

http://www.tasciences.com/what-is-a-telomere/
 Immortalised cell lines

• Cells cultured from hosts and treated to

multiply indefinitely
• Mutated or genetically altered
• Commercialised cell lines for research
– Allows for repeatability and consistency
– Commonly used
• Hela
• HaCat
 HeLa cells
• Human cervical cancer cells
• Isolated from patient : Mrs Henrietta Lacks
• Biopsy taken 080251, patient died 041051
• Characteristics
– First type of human cells that were successfully
cultivated in vitro outside of human body
– Cells proliferate abnormally
• rapid - even compared to other cancer cells
– Have active telomerase
Lacks’ story has previously been the subject of an award-winning
documentary, The Way of All Flesh, directed by Adam Curtis.
http://www.kontrolmag.com/oprah-winfrey-star-henrietta-lacks-movie/
 Contamination
• Source
– Individuals
– Environment
– Equipment
– Consumables
• Physical detection
– Viewing link
• Chemical detection
Link to Recommended Reading
 Cell line cross contamination

• Misidentification
• Detection
• Authentication
– Recommended reading
– Dunham & Guthmiller 2008
 Bacterial contamination

• Types
– Rods (bacilli)
• 1 m diameter x 4 m length
– Round (cocci)
• 1 m
– Spiral (spirilla)
• 0.5-1.0 m diameter x 10-20 m length
• Features
 Fungal contamination

• Types
– Mycelium
– Spores
• Features
– Seasonal
– Longevity
Yeast contamination

GIBCO 2013
 Mycoplasma contamination

• Bacterial size = 1 m
• Sources
• Affects
– Chromosomes
– Metabolism
– Cell growth
• Testing
• Treatment
26
27
Bionique 2011
 Which is a clean culture?

1. Left
2. Middle
3. Right
4. All
5. None
 Transformation of Cells
• Change in the phenotype of cells
– Immortalisation:
• acquisition of infinite life span
• cells grow beyond Hayflick limit
• Cells overcome senescence

– Aberrant growth control:

• loss of contact inhibition of cell motility,
• density limitation of cell proliferation
• No anchorage dependence

– Aberrant growth control:

• Growth of invasive tumour in vivo
 Transformation of Cells
– Spontaneous transformation
• Cells bypass senescence

– Induced transformation
• Virus induced
SV40 large T antigen,
HPV,
EBV
• Telomerase induced
– hTERT transfections
Telomerase transfection

http://www.tasciences.com/what-is-a-telomere/
 Cryopreservation (Storage):
Why?
• Backup for contamination

• Senescence in continuous cultures

• Genetic instability

• Phenotype instability

• To avoid cross contamination and mis-identification

• Saving time and cost in maintenance

• Distribution to other users

How?
 Ground rules for CP
• Slow cooling (1oC/ min)
– allows water to leave the cells
• Not too slow
– ice crystal formation leads to cryogenic damage
• Hydrophilic Cryoprotectant (e.g. DMSO, Glycerol etc)
– sequesters water
• Ultralow temperature storage (LN2)
– minimizes the effect of high salt concentration on
proteins within the cells
• Rapid thawing
– minimizes the ice crystal growth
– establishment of solute gradients as ice melts
 Application 1:
Drug Discovery & In vitro Toxicity

NCI-60 DTP Human Tumour Cell Line Screen

http://dtp.nci.nih.gov/branches/btb/ivclsp.html
 MTT Assay
Principle:
• A colorimetric assay that measures the reduction of yellow 3- (4, 5-
dimethythiazol- 2- yl)-2, 5- diphenyl tetrazolium bromide (MTT) by
mitochondrial dehydrogenase enzymes, forming insoluble formazan
(purple) crystal. The formazan crystal are then dissolved with
acidified organic solvent and measured spectrophotometrically
between 570-600 nm.

Formazan Crystal
 70-80% confluent flask with cells were trypsinized and adjusted to the final
volume of 5,0000 to 100,000 cells/ml

Cells seeded @ density of 5,000 to 10,000 per well i.e. 100 μL in each well
Assay Procedure

Incubation at 37°C and 5% CO2, Overnight/16 hours

Cells were treated with desired concentrations of drug/Nanoparticle

Incubation at 37°C and 5% CO2, for 24-72 hours

Cells incubated for 4 hours until purple precipitate is visible

100-200 μL of Acidified isopropanol was added per well

Readings recorded at absorbance 595 and 630 nm

MTT Assay
Representation of Plate design to determine the plate toxicity
1 2 3 4 5 6 7 8 9 10 11 12
A
B 0
C 1
D 2.5
E 5
F 10
20
G
H

No Drug
Measurement count: 1 Filter: 595
Increasing concentration of drug
1 2 3 4 5 6 7

0 0.647 0.645 0.638 0.649 0.006 0.07

1 0.605 0.591 0.598 0.59 0.016 0.018
2.5 0.5 0.519 0.504 0.515 0.014 0.015
5 0.4 0.432 0.47 0.46 0.012 0.012
10 0.345 0.348 0.36 0.331 0.019 0.014
20 0.214 0.208 0.246 0.229 0.019 0.019
Calculation

Test OD (Av) – Blank OD(Av)

% Viability = Control OD (Av) – Blank OD(Av)
X 100
 Dose and time dependent toxicity of Test Drug
on Prostate Cancer Cells (PC-3)
140.00

120.00

100.00
% Viability

80.00
24 hours
60.00 72 hours

40.00

20.00

0.00
0 25 50 100 125 150
Concentrations (μM)

Fig 2. Dose and time dependent toxicity of tested drug on PC-3 cells. Cell viability was
determined by MTT assay and reported as percentage of viable cells relative to control

Advantages and limitations of MTT assay

 Advantages and limitations of
MTT assay
• Advantages
– Colorimetric and cost effective
– Reliable for cell viability,
– High throughput

• Limitations
– Chemical Interference
– Not a direct assay for cell numbers
– Does not tell the growth phase of cells