Download Virus isolation in traditional and newer cell cultures

‫بسم اهلل الرحمن الرحيم‬
‫”‬
‫صدق اهلل العظيم‬
‫(أل عمار‪ :‬ايه ‪)8‬‬
Virus isolation in traditional cell cultures
and newer cell culture formats
By
Wafaa Kamel Mowafy
Prof. of Microbiology and Immunology , Mansoura
Faculty of Medicine
• The discovery in the early 1900s that human cells could
be propagated in vitro provided virologists with an
alternative to embryonated eggs and laboratory animals
for in vitro isolation of viruses. By the early 1970s,
diagnostic virology expanded dramatically, largely
because of the availability of highly of purified reagents
and commercially prepared cell lines.
Virus isolation in traditional cell cultures
(monolayer cultures)
1. Primary culture
2. Semi -continuous cell culture
3. Continuous cell culture
Cell monolayers are most commonly used for
culture of viruses. The are three categories, namely
(1) primary, (2) semi continuous, and (3) continuous
cell cultures.
• Primary cultures. Viable cell suspensions may be
obtained by dissociating tissues or organs, e.g.
human amnion, with trypsin, collagenase or other
enzymes.
Advantages .
Disadvantages.
• Semi continuous cell cultures (cell strains )
– Semi continuous cell cultures are established with the
successful subculture of primary cell monolayers.
These cultures consist mostly of spindle shaped
fibroblast cells. Established from human embryonic
tissue, or neonatal foreskin. .
– Advantages.
– Disadvantages.
• Continuous cell cultures ( cell lines ).
Continuous cultures are produced either by
transformation ( spontaneous or engineered ) of cell
strains in vitro, or by culture of cells taken from tumors
e.g Hela ( human cervical carcinoma ) and a human
rhabdomyosarcoma cell line (RD cells ).
– Advantages.
– Disadvantages.
The following continuous cell lines are commonly used:
 Hela and HEp2 are used for cultivation of HSV,
adenovirus, poliovirus and some coxsackie viruses. Vero
cells will also support growth of these viruses and are
used with BHK21 cells for growth of arboviruses.
 RK13 cells and BHK21 cells for isolation and
propagation of rubella virus . RD cells for the isolation of
coxsackie A virus .
• Newer cell culture formats
1. Centrifugation-enhanced inoculation and pre-CPE
detection of viruses in cell culture.
2.
Virus isolation in transgenic cell lines
3.
Virus isolation in cocultured cells.
– Centrifugation-enhanced inoculation and pre-
CPE
detection of viruses in cell culture.
 Cytospinning ( shell vials ). In this assay cells are
grown on a coverslip in a vial , infected with the
specimen and submitted to low- speed centrifugation
followed by over night incubation. Labeled antibody
to early antigen is used to stain cells after 24- 48
hours.
Figure 5.17a: Tissue culture cells are
grown on coverslips on the bottom of
shell vials.
Reproduced from Athmanathan, S., S. R. Bandlapally, and G. N. Rao, BMC Clin. Pathol.
2 (2002): 1-5.
Figure 5.17b: Detection of Herpes
Virus Simplex 1 using the shell vial
technique and immunofluorescence.
Modified from J. H. Shelhamer, et. al., Ann. Intern. Med. 124 (1996): 585-599.
Shell vial technique
• Centrifugation Culture (Shell Vial
Technique)
– Used a lot in clinical labs
• Engineered cell lines (Enzyme-linked virus-
inducible system , Transgenic cell lines)
• The application of transgenic cells in cell cultures
involves the stable introduction of genetic elements into
a cell such that when a virus, and only a particular virus,
enter this cell ,a virus specific event is trigged that results
in the production of an easily measurable enzyme.
 ( baby hamster kidney – inducible B -galactosidase gene
with HSV – inducible promoter ) was designed for sensitive
and specific detection of both HSV-1 and HSV-2.
ً
When these cells are infected with either HSV-1 or HSV-2
, the B - galactosidase is induced and then an X- gal
colorimetric substrate is added, infected cells turn blue,
while other viruses do not induce the enzyme and the cells
remain colorless.
• This method has been incorporated into a commercial kit
that has been marketed as the enzyme – linked virusinducible system{ ELVIS}.
• There are advantages to this method including its
rapidity, use of relatively inexpensive substrate as
compared to antibodies, and the ability to use cells to
look for both the color changes and CPE. The method is
adaptable for other viruses, both DNA and RNA viruses.
• Virus isolation in cocultered cells
• Techniques involving combination of different cell types
grown together as a single monolayer in a vial and the
application of various MAbs, each labeled with a different
fluorochrome for the detection of several viruses in the
same vial.
• .
– The choice of cells has been focused on the ability to
isolate and identify viruses that have a common
pathological presentation, such as enteroviruses or
respiratory viruses.
– . An example of This, R- mix uses a combination of
A549 cells and mink lung cells and is useful for the
detection of adenoviruses, parainfluenza viruses,
andٌ RSV. Three R- mix vials are inoculated for each
specimen.
Preliminary results using these cell mixtures indicate
that they perform similarly to or better than cell culture
or single – cell shell vial cultures and are more cost
effective .
• Selection for culture media
A range of media have been formulated for growth of
vertebrate cells in culture. These incorporate various
conc. Of amino acids, vitamins, enzymes, growth factors,
and inorganic salts. Glucose, fructose, or galactose are
also added along with glutamine to provide a carbon
source for cell metabolism.
• There is a variety of formulations for cell culture media:
– Dulbecco,s Minimal Essential Medium (DMEM) is in
common use for continuous cell lines.
– CMRL medium is particularly suited for the
propagation of semi -continuous cell lines.
– RPMI 1640 is recommended for growth of
Lymphoblastoid cells in suspension.
• Conditions for growth of cell cultures
• Optimum pH. A pH range of 7.1-7.5 is required for the
growth of eukaryotic cells. Most culture media use
bicarbonate buffer systems (co2 / Hco 3) to maintain Ph.
These media are formulated with NaHco 3and Co2 is
either provided by the cultured cells as a metabolic
product or by enrichment of the atmosphere using a co 2
incubator.
• It is common to supplement the bicarbonate buffer
system with HEPES buffer , it overrides all other buffers
present and obviates the need for co2-enriched
atmosphere.
• Osmolarity. The growth of cells in culture depends on
an optimum range of osmotic pressures, usually
between 280 and 320 mmol/kg.
• Serum. Balanced salt solutions will support cell
proliferation only when supplemented with serum,
lactalbumin hydrolysate, or other supplements.
The serum has several functions :
 It provides essential amino acids, nucleic acid
precursors, and fatty acids.
 It provides hormones and inhibits the protease used for
routine dissociation of cells for culture.
• Fetal or new – born calf serum are used at conc. 5-15 %
to promote cell growth and at reduced conc. Of 0-2 % for
maintenance of confluent monolayer cultures.
• Serum should be stored at –70 C , repeated freezing and
thawing should be avoided.
• Antibiotics. antibiotics providing broad spectrum
protection from bacterial contaminants are :
benzylpencillin, 20-100 units/ml, gentamicin, 16-50 ug /
ml, and tetracycline, 10 ug/ ml.,Amphotericin B, 0.5 ug/
ml, or nystatin, 50 units/ ml. are recommended for
control of fungal infections.
• Stock solutions should be stored at –20 C .
•
Subculture of semi-continuous or continuous
cell cultures ( method -1 )
1.
Pour off culture medium and wash the cell sheet twice
with phosphat buffered saline(PBS)
2.
Add sufficient amount of trypsin-EDTA solution to
cover cells.
3.
Incubate at room temperature until cell sheet appears
opaque. At this stage the cells will be rounded but not
detached when observed with an inverted microscope.
This process usually takes 1-3 min.
• 4 .Remove excess trypsin solution. Cells will be
detached from the culture vessel after approximately 2-3
min
• 5. Add a small amount of chilled growth medium and
aspirate several times with a 10 ml pipette to suspend
and separate cells.
• 6. Dilute a small sample of the cell suspension with
additional growth medium for cell counting or dispense
directly into new growth vessels. Semi -continuous
fibroblast are generally passed, one-to-two split. A one –
to-six up to a one-to-ten split is common for continuous
cultures.
• 7. When the monolayer reaches confluence, the growth
medium should replaced with maintenance medium ( 2%
serum).
• Method -2
• 1-2 as method-1
• 3. Incubate the cells until cell detachment occurs.
• 4. Add about 10 ml of growth medium , mix and transfer
to centrifuge tube. Centrifuge for 10 min. at 1000rpm,
remove supernatant and resuspend pellet in growth
medium.
• 5,6 and 7 steps as method 1.
• Determination of cell numbers and viability by
haemocytometer:
• Disperse cells and dilute o.1ml of cell suspension with
o.9 ml trypan blue solution in a separate container.
• Moisten the supporting ridges of the haemocytometer
chamber and apply a cover –slip.
• After 4-5 min in trypan blue solution , resuspend cells
and fill both sides of the counting chamer without
overflowing.
• Count the number of cells in the four corner squares and
in the center of each side (10 squares).
• Calculate the number of cells in the original suspension.
Each squar represents an area of.1mm2 and has a depth
of o.1 mm therefore the volume held by each squar is
o.1 mm3 and the sample volume is 1o x o.1 or 1 mm3.
To determine the cell conc.of the original flask the
following formula is used:
• Number of cells counted x 10 ( dilution factor) x
1000(no. of mm3 in cm3 = number of cells /ml. for
example when 250 cells counted in 10 squars , the cell
conc. in the original solution 250x10x1000 =2.5x106
cells/ ml.
• To determine viability , make separate counts of both
stained ( dead) and non stained (live) cells in a given
area .A total of at least 100 cells should be counted.