Characterization of Cell Line

Cell line: Once a primary culture is sub-cultured
or passaged
Normal cell line: Divides a limited number of
times
Continuous cell line: Cell line having the capacity
for infinite survival (Immortal)
Characterization is the defining/ outlining those
many traits of the cell line………some of which
may be unique !

The Need !!
♟Authentication, i.e., confirmation that the cell
line is not cross-contaminated or misidentified
♝Confirmation of the species of origin
♞Correlation with the tissue of origin, which
comprises the following characteristics:
♛Identification of the lineage to which the cell
belongs
♚Position of the cells within that lineage (i.e., the
stem, precursor, or differentiated status)

♟Determination of whether or not the cell line is
transformed:
❶Is the cell line finite or continuous
❷Does it express properties associated with
malignancy
♜Indication of whether the cell line is prone to
genetic instability and phenotypic variation
♞Identification of specific cell lines within a
group from the same origin, selected cell
strains, or hybrid cell lines, all of which require
demonstration of features unique to that cell
line or cell strain

♫ The proper use of a cell line (whether in research or
commercial exploration) requires that they are validated
♫ Industrial- environment- legal obligation (FDA)
♫ Academic research laboratory: less well defined and the
obligation left to individual consciences

♫ Provenance:

What has happened to the cell line since its original
isolation?
Records detailing the origin, characteristics, and
handling of the cell line form the provenance of the
cell line
The more detailed the provenance, the more
valuable the cell line

Criterion/ Method for Characterization…

CELL MORPHOLOGY

(Freshney, 2010)

Nature of technique depends on type of work
☻ If molecular technology is readily available, then
DNA profiling or analysis of gene expression are
likely to be of most use
☻ A cytology laboratory may prefer to use
chromosome analysis coupled with FISH and
chromosome painting
☻ A laboratory with immunological expertise may
prefer to use MHC analysis (e.g., HLA typing)
coupled with lineage specific markers
☻ Combine with a functional assay related to your
own interests

CELL MORPHOLOGY
♥ Study of the size, shape, and structure of cell.
♥ Most cells in culture can be divided in to five basic
categories based on their morphology.
♠ Fibroblastic/ Fibroblastoid (Fibroblast-Like)
♠ Epithelial/ Epithelioid(Epithelial-Like)
♠ Lymphoblast-Like
♠ Endothelial
♠ Neuronal

Confluency
♞Confluency is the term commonly used as a
measure of the number of the cells in a cell
culture dish or a flask, and refers to the
coverage of the dish or the flask by the cells
♞For example, 100 percent confluency means
the dish is completely covered by the cells, and
therefore no more room left for the cells to
grow
♞50 percent confluency means roughly half of
the dish is covered and there is still room for
cells to grow.

∗ Snu449 at 50-60
per cent confluency

∗ Snu449 at 100 per
cent confluency

The initial exponential growth of the culture is followed by a plateau
phase when cells reach confluence

CELL MORPHOLOGY
♥Observation of morphology:
ᴥ Simplest and most direct technique to identify cells
ᴥ Shortcomings- related to the plasticity of cellular
morphology in different culture conditions
ᴥ Epithelial cells growing in the centre of a confluent
sheet are regular, polygonal & with clearly defined
birefringent edge
ᴥ The same cells growing at the edge of a patch may be
more irregular and distended and
ᴥ if transformed, may break away from the patch and
become fibroblast-like in shape

Subconfluent: spatial relationships of the
cells are intermediate between sparse and
confluent

start to form multilayers

Cells that have changed or
have been changed in a way
that differs to their normal
counterparts..

∗ Subconfluent fibroblasts from hamster kidney
or human lung or skin assume multipolar or
bipolar shapes (see Fig. a, g) and are well spread
on the culture surface,
∗ but at confluence they are bipolar and less well
spread (see Fig. b, h). They also form
characteristic parallel arrays and whorls that are
visible to the naked eye

∗ Mouse 3T3 cells (Fig. s, t) grow like multipolar
fibroblasts at low cell density

∗ But Mouse 3T3 cells become epithelioid at confluence
(Fig. v, w).

∗ Alterations in the substrate and the
constitution of the medium can also affect
cellular morphology
∗ Comparative observations of cells should
always be made at the same stage of growth
and cell density in the same medium, and for
growth on the same substrate

∗ The terms ‘‘fibroblastic’’ and ‘‘epithelial’’ are
used rather loosely in tissue culture and often
describe the appearance rather than the origin of
the cells
∗ Thus a bipolar or multipolar migratory cell,
whose length is usually more than twice its
width, would be called fibroblastic
∗ whereas a monolayer cell that is polygonal, with
more regular dimensions, and that grows in a
discrete patch along with other cells, is usually
regarded as epithelial

“When the identity of the cells has not been
confirmed, the terms ‘‘fibroblast-like’’ (or
‘‘fibroblastoid’’) and ‘‘epithelium-like’’ (or
‘‘epithelioid’’) should be used”

∗ Carcinoma-derived cells are often epithelium-like
but more variable in morphology (Fig. 15.2m, n, k, l)

∗ Some mesenchymal cells like CHO-K1 and
endothelium can also assume an epithelium-like
morphology at confluence (Fig.d, r)

Fibroblastic (or fibroblast-like) cells are bipolar or
multipolar, have elongated shapes (length usually more
than twice the width), and grow attached to a substrate

Epithelial-like cells are polygonal in shape with more
regular dimensions, and grow attached to a substrate in
discrete patches

∗ Lymphoblast-like cells are spherical in shape and
usually grown in suspension without attaching to a
surface

Endothelial cells are very flat, have a central nucleus, are
about 1-2 µm thick and some 10-20 µm in diameter
Human pulmonary microvascular endothelial
cells (HPMEC) cultures after cotransfection
with plasmids encoding human telomerase
reverse transcriptase protein (hTERT) and
simian virus 40 (SV40) large T antigen.
Morphology of HPMEC-ST1.6R cells after 1
year of continuous propagation. A confluent
monolayer exhibiting convex polygonal
shape of the cells (A), at preconfluent stage
(B)

Citation:
“Generation of Human Pulmonary Microvascular Endothelial Cell Lines”
Laboratory Investigation, Nature Publishing group
Authors: Vera Krump-Konvalinkova, Fernando Bittinger, Ronald E Unger, Kirsten Peters,
Hans-Anton Lehr and C James Kirkpatrick

Neuronal cell line
Exist in different shapes and

sizes, but they can roughly be
divided
into
two
basic
morphological categories,
Type I with long axons used to
move signals over long distances
and
Type II without axons

∗ Precursor cells that are still capable of diving are
called blast cells: for example,
∗ a fibroblast is a proliferative precursor of a
fibrocyte
∗ a myoblast is a proliferative precursor of a
myocyte
∗ a lymphoblast is a proliferative precursor of a
lymphocyte
“These rules of terminology are not always
observed”

Phase contrast images of healthy 293 cells in adherent culture
10X and 20X objectives (panels A and B, respectively)

Phase contrast images of healthy 293F cells grown is suspension

Cell line

Meaning

Organism

Origin tissue

Morphology

BEAS-2B

Bronchial
epithelium +
Adenovirus 12-SV40
virus hybrid
(Ad12SV40)

Human

Lung

Epithelial

BHK-21

"Baby Hamster
Kidney Fibroblast
cells"

Hamster

Kidney

Fibroblastic

HL-60

Human leukemia

Human

Myeloblast

Bloodcells

MDCK II

Madin Darby canine
kidney

Dog

Kidney

Epithelium

CHROMOSOME CONTENT
♚Chromosome content or karyotype is one of the most
characteristic and best-defined criteria for identifying
cell lines and relating them to the species and sex from
which they were derived
♚Karyotype:
systematic, ordered representation of the entire
chromosome of a cell
number and appearance of chromosomes in the
nucleus of a eukaryotic cell
describe the number of chromosomes, and what they
look like under a light microscope

Stage

chromosome number, sex /
chromosomes,aberrations

normal female/male

46,XX / 46,XY

klinefelter-syndrome

47,XXY

turner-syndrom (monosomy X)

45,X

trisomy 21, male

47,XY,+21

translocation

46,XX,t(9;22)(q11;q34)

deletion

46,XX,del(2)(q23q32)

∗ Shows the chromosomes as they appear in metaphase

 Karyotype : An orderly display of magnified
images of the individual’s chromosomes
 The chromosomes are depicted in a standard
format known as a karyogram or ideogram: in
pairs, ordered by size and position of
centromere for chromosomes of the same size.
 Karyotype analysis:
chromosomes
are
microscope.

a technique where
visualized
under
a

Ideogram:
∗Diagrammatic representation of
the gametic chromosome set (n)
of a species
∗Used to compare the karyotype
of one species with the other
∗bands
locate
sites
on
chromosome
Shown only
one set of
chromosomes

♥Karyotypes are presented
☻By arranging chromosomes of somatic
complement in a descending order of size
keeping their centromeres in a straight line
☻Longest chromosome – on extreme left
☻Shortest chromosome – on extreme right
☻Sex chromosomes – allosomes – extreme right

Is this Male or Female Karyotype?

Down Syndrome Karyotype

∗ Trisomy 21

♞

Karyotype analysis is best criteria for species
identification

♞ Genetic stability of ES and iPS cells are routinely
monitored by karyotype analysis
♞ Normal and transformed cells can be distinguished
♞ Comparative phylogenetic studies of two species can
be done
♞ Confirmation or exclusion of a suspected crosscontamination

Karyotype of ES cell lines. G-band analysis of karyotypes of KhES-1,
-2, and -3 at passages 245, 177, and 177, respectively. KhES-1 and
KhES-2 had a female karyotype, and KhES-3 had a male karyotype.
KhES-3 cell line showed normal karyotyp...

Hirofumi Suemori , Kentaro Yasuchika , Kouichi Hasegawa , Tsuyoshi Fujioka , Norihiro Tsuneyoshi , Norio Nakatsuji
Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage
Biochemical and Biophysical Research Communications Volume 345, Issue 3 2006 926 - 932
http://dx.doi.org/10.1016/j.bbrc.2006.04.135

∗ Chromosome analysis can also distinguish
between normal and transformed cells because
the chromosome number is more stable in
normal cells (except in mice)

Chromosome Preparation
A. Metaphase
Block

Duration of the metaphase block may
be increased to give more metaphases
for
chromosome
counting,
or
shortened to reduce chromosome
condensation and improve banding

B. Collection
of Mitosis &
C. Hypotonic
Treatment

Chromosome Banding
∗ Devised to enable individual chromosome pairs to be identified
when there is little morphological difference between them

“Treatment of chromosomes to reveal characteristic
patterns of horizontal bands is called chromosome
banding.”
The banding pattern lend each chromosome a
distinctive appearance.
Banding also permits recognition of chromosome
deletions, duplications and other types of structural
rearrangements of chromosomes.

Types
G–Banding:
∗ Staining a metaphase chromosome with Giemsa stain
is called G-Banding.
∗ preferentially stains the regions that are rich in
adenine and thymine and appear dark.
C-Banding:
Specifically stain the centromeric regions and other
regions containing constitutive heterochromatin.

Q-Banding
 Quinacrine mustard (a fluorescent stain), an
alkylating agent, was the first chemical to be used
for chromosome banding
 Quinacrine bright bands were composed primarily
of DNA rich in bases adenine and thymine
Used to identify

specific
chromosomes
and
structural
rearrangements
 various polymorphisms involving satellites and
centromeres of specific chromosomes

R (reverse banding)
 R-banding is the reverse of G-banding.

The dark regions are euchromatic (guaninecytosine rich regions) and the bright regions are
heterochromatic (thymine-adenine rich regions).
T-banding: visualize telomeres
NOR (nucleolar organizing regions)

Silver nitrate stains selectively the satellite
stalks of the acrocentric chromosomes.

a) C-banding b) R-banding c) Q-banding d) G-banding

Brief notes……….
∗ For Giemsa banding, the chromosomal proteins are partially
digested by crude trypsin, producing a banded appearance on
subsequent staining.
∗ Trypsinization is not required for quinacrine banding. The
banding pattern is characteristic for each chromosome pair
∗ Other methods for banding include the following:
Giemsa banding using trypsin and EDTA rather than trypsin
alone
Q-banding, which stains the cells in 5% (w/v) quinacrine
dihydrochloride in 45% acetic acid, followed by rinsing the slide,
and mounting it in deionized water at pH 4.5
C-banding, which emphasizes the centromeric regions

∗ Techniques have been developed for
discriminating between human and mouse
chromosomes, principally to aid the karyotypic
analysis of human-mouse hybrids.
∗ These methods include fluorescent staining
with Hoechst 33258, which causes mouse
centromeres to fluoresce more brightly than
human centromeres and alkaline staining with
Giemsa (‘‘Giemsa-11’’)

CHROMOSOME PAINTING
“Rendering a specific chromosome or chromosome segment
distinguishable by DNA hybridization with a pool of many
fluorescence-labeled DNA fragments derived from the full length of a
chromosome or segment is called chromosome painting”
(McGraw-Hill Dictionary)
 This technique employs in situ hybridization technology, also
used for extra chromosomal and cytoplasmic localization of
specific nucleic acid sequences like specific mRNA species
 SKY and M-FISH are newer karyotyping methods based on
chromosome painting techniques…allow the simultaneous
visualization of all 23 human chromosomes in different colors

∗ Chromosome paints are available commercially from a
number of sources

SKY is a powerful ,whole-chromosome painting assay that
allows the simultaneous visualization of each
chromosome in different colors

Five spectrally distinct dyes are used in combination to
create a cocktail of probes unique to each chromosome

 The probe mixture is hybridized to metaphase
chromosomes on a slide and then visualized with a
spectral interferogram cube, which allows the
measurement of the entire emission spectrum with a
single exposure
The image is processed by computer software that
can distinguish differences in color not discernible to
the naked eye by assigning a numerical value to the
RGB

Spectral karyogram of a human female
(Schrock et al., 1996)

SKY can detect
Chromosomal material of unknown origin,
complex rearrangements, translocations, large
deletions, duplications, aneuploidy

Disadvantages
Ineffective detection of micro deletions and
inversions
 It can only be performed on dividing cells

Multicolor fluorescence in situ
hybridization (M-FISH)
It is based on chromosome painting
M-FISH identifies translocations and insertions
Reliable tool for diagnostic applications and Interphase
nuclei are hybridized with the FISH probe
M-FISH is filter-based technology which does not rely
on specialized instrumentation for its implementation as
SKY

Characterization of structural rearrangements: M-FISH (multicolor FISH) is used to detect
a complex chromosome rearrangement involving a translocation between chromosome
6 and 16, as well as between chromosomes 2 and 10.

Chromosome Analysis
Methods:
(1) Chromosome count: Count the chromosome number
per spread for between 50 and 100 spreads. (The chromosomes
need not be banded.) Closed-circuit television or a camera lucida
attachment may help. You should attempt to count all of the
mitoses that you see and classify them
(a)by chromosome number or, if counting is impossible,
(b) as ‘‘near diploid uncountable’’ or ‘‘polyploid uncountable.’’
Plot the results as a histogram

(2) Karyotype:
Digitally photograph about 10 or 20 good spreads of
banded chromosomes
Using Adobe Photoshop or an equivalent graphics
program, cut the individual chromosomes and paste
them into a new file where they can be rotated, trimmed,
aligned, and sorted
Image analysis can be used to sort chromosome images
automatically to generate karyotypes (e.g., Leica
CW4000)

∗ Chromosome counting and karyotyping allow
species identification of the cells and, when
banding is used, distinguish individual cell line
variations and marker chromosomes
∗ However, karyotyping is time-consuming, and
chromosome counting with a quick check on
gross chromosome morphology may be
sufficient to confirm or exclude a suspected
cross-contamination

It involves three methods:

DNA hybridization
DNA fingerprinting
DNA profiling

DNA CONTENT
DNA content can be measured by using DNA
flourochromes:
 Propidium iodide
 Hoechst 33258
 DAPI
 Pico green
Analysis of DNA content is particularly useful in the
characterization of transformed cells that are often
aneuploid and heteroploid

Provide information about :
♛Species-specific regions.
♛Amplified regions of the DNA
e.g. amplification of DHFR (Dihydrofolate reductase)
gene, in cell lines selected for resistance to methotrexate
♛ Altered base sequences that are characteristic to that
cell line.
e.g. Over expression of a specific oncogene in
transformed cell lines

☻ Technology using Variable number of tandem

repeats present in genome to identify individual
cells
☻ DNA contains regions known as satellite DNA that
are apparently not transcribed
☻ They give rise to regions of hyper variability
☻ Cross contamination is confirmed by it

The following techniques are used for DNA
fingerprinting analysis :
 RFLP (Restriction Fragment Length Polymorphism)

AmpFLP
(Amplified
Fragment
Length
Polymorphism)
 STR (Short tandem repeats)
 SNP (Single Nucleotide Polymorphism)

.
D A Gilbert et.al “Application of DNA fingerprints for cell-line individualization”Am J Hum
Genet. 1990 September; 47(3): 499–514.

DNA profiling primarily examines "short tandem repeats,"
or STRs.
STRs are repetitive DNA elements between two and six
bases long that are repeated in tandem

These STR loci are targeted with sequence-specific primers
and amplified using PCR.
Most extensively used with human cell lines.

∗ From country to country, different STR-based DNAprofiling systems are in use
∗ In North America, systems which amplify the CODIS 13
core loci are almost universal
∗ In the UK the SGM+ system is in use
∗ SGM examines 7 (loci) different areas of the genome,
where as SGM PlusTM examines 11
∗ DNA profiling is available as a service from a number
of laboratories including LGC and ATCC

Bovine genotype encompasses the following eighteen STR
loci:
 TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122,
INRA23, ETH3, ETH225, BM1824 BM1818,SPS113, RM067,
CSRM60, MGTG4B, CSSM66 and ILSTS006.
 These are among the list of loci recommended by the
International Society for Animal Genetics(ISAG) and the Food
and Agriculture Organization of the United Nations (FAO).

☻ DNA profiling has been used most extensively
with human cell lines where the primers are
most commonly available and the extension of
this to other animal species is still somewhat
limited
☻ Speciation can be achieved however using the
so-called “barcode region’’ of the cytochrome
oxidase I as well as by isoenzyme analysis

“Characterization of cell line is the first
indispensible step after each cell line is
generated for determining its functionality,
authenticity, contamination ,origin etc”
“Morphology, Chromosome and DNA analysis
have now became the major standard
procedure for cell line identification”

REFERENCES
D A Gilbert et.al “Application of DNA fingerprints for
cell-line individualization”Am J Hum Genet. 1990
September; 47(3): 499–514
Freshney R.Ian, “Culture of Animal Cells: A Manual of
Basic Technique”(2010) :239-260
J.G.Dauwerse, J.Wiegant, A.K.Raap, M.H.Breuning and
G.J.B.van Ommen “Multiple colors by fluorescence in
situ hybridization using ratio-labelled DNA probes
create a molecular karyotype” Human Molecular
Genetics, Vol. 1, No.8: 593-598

∗ Thomas Ried, Evelin Schlock, Yi Ning and Johannes
Wienberg “Chromosome painting: a useful art” Human
Molecular Genetics, 1998, Vol. 7, No. 10 Review 1619–1626.
∗ Kamila Schlade-Bartusiaka, Maria M. Sasiadeka,Julia K. Barb,
Steffi Urbschatc,Nikolaus Blind, “Cytogenetic and molecular
cytogenetic characterization of the stable ovarian
carcinoma cell line (OvBH-1)” Cancer Genetics and
Cytogenetic 164 (2006) 10–15.
∗ And the list is long……………

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