INTRODUCTION

Making Sense!!!

Details not clear??

What about now !!

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 !

♟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)

The Need !!

♟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…

(Freshney, 2010)

CELL MORPHOLOGY

☻ 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

Nature of technique depends on type of work

CHAPTER I

♥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

CELL MORPHOLOGY

♞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.

Confluency

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

♥Observation of morphology:ᴥSimplest and most direct technique to identify

cellsᴥShortcomings- related to the plasticity of

cellular morphology in different culture conditions ᴥEpithelial cells growing in the centre of a

confluent sheet are regular, polygonal & with clearly defined birefringent edge

ᴥThe 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

CELL MORPHOLOGY

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 groupAuthors: 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-60Human

leukemiaHuman Myeloblast Bloodcells

MDCK IIMadin Darby canine kidney

Dog Kidney Epithelium

CHAPTER 2

♚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 cellnumber and appearance of chromosomes in the nucleus of a eukaryotic celldescribe the number of chromosomes, and what they look like under a light microscope

CHROMOSOME CONTENT

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: a technique where chromosomes are visualized under a microscope.

Ideogram: Diagrammatic representation of the gametic chromosome set (n) of a speciesUsed to compare the karyotype of one species with the otherbands 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

Normal Human Male Karyotype

Normal Human Female

Karyotype

Is this Male or Female Karyotype?

Down Syndrome Karyotype

Trisomy 21

Normal Karyotype in

Cattle

♞ 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 cross-contamination

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 NakatsujiEfficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passageBiochemical and Biophysical Research Communications Volume 345, Issue 3 2006 926 - 932http://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 PreparationDuration of the metaphase block maybe increased to give more metaphases for chromosome counting, or shortened to reduce chromosome condensation and improve banding

A. Metaphase

Block

B. Collection of Mitosis &

C. Hypotonic Treatment

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

Chromosome Banding

“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.

TypesG–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.

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

Q-Banding

R (reverse banding) R-banding is the reverse of G-

banding. The dark regions are euchromatic

(guanine-cytosine 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

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 aloneQ-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

Brief notes……….

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’’)

(A) (B)

(C)

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

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

Multicolor fluorescence in situ hybridization (M-FISH)

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.

Methods:

(1) Chromosome count: Count the chromosome numberper 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

Chromosome Analysis

(2) Karyotype:

Digitally photograph about 10 or 20 good spreads of banded chromosomesUsing 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 sortedImage 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

CHAPTER 3

It involves three methods:

DNA hybridizationDNA fingerprintingDNA profiling

DNA CONTENTDNA 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 DNA-profiling 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……………

Thank You