Post on 16-Feb-2017
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INTRODUCTION
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
♫ 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…
☻ 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
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 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
BHK-21 (baby hamster kidney fibroblasts)
Sub confluent fibroblasts from hamster kidney or human lung or skin assume multipolar or bipolar shapes and are well spread on the culture surface, but at confluence they are bipolar and less well spread They also form characteristic parallel arrays and whorls that are visible to the naked eye
HEK293human embryonic kidney epithelial cell line
LNCaP clone FGC from a lymph node metastasis of prostate carcinoma
HeLa cells from human cervical carcinoma
CHO-K1 cloned line of Chinese hamster ovary
Vero cells in log phase;
MRC-5 human fetal lung fibroblasts
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”
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
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
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
hybrid
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
CHAPTER 2
♚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)
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
♥Karyotype : An orderly display of magnified images of the individual’s 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 cells are routinely monitored by karyotype analysis
♞ Normal and transformed cells can be distinguished
♞ 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 abormal karyotype at 16th chromosome.
Chromosome analysis can also distinguish between normal and transformed cells because the chromosome number is more stable in normal cells.
Chromosome abberations
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
Dropping suspension on to a slide and air dried
staining with
giemsa and cover
slip applied
examine under oil
immersion objective
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.
when there is little morphological difference between them
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 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: G 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……….
CHROMOSOME PAINTING“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”
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 chromosomes in different colours
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 The image is processed by computer software that can distinguish differences in color which naked eye cannot, by assigning a numerical value to the RGB
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
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.)
(2) Karyotype: Digitally photograph about 10 or 20 good spreads of banded chromosomesUsing Photoshop cut the individual chromosomes and paste them into a new file where they can be rotated, trimmed, aligned, and sorted
Chromosome Analysis
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-(4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA 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 ♛ Altered base sequences that are characteristic to that cell line. e.g. Over expression of a specific oncogene in transformed cell lines
☻ Technology using (VNTR) Variable number of tandem repeats present in genome to identify individual cells
☻ DNA contains regions by it☻known as satellite DNA that are apparently not transcribed
☻ Cross contamination is confirmed
☻Variations of VNTR (D1S80) allele lengths in 6 individuals.
The following techniques are used for DNA fingerprinting analysis :
RFLP (Restriction Fragment Length Polymorphism)
AmpFLP (Amplified Fragment Length Polymorphism)
STR (Short tandem repeats)- containing core repeat units of between two and seven nucleotides
SNP (Single Nucleotide Polymorphism)
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.
☻ 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
REFERENCES
Freshney R.Ian, “Culture of Animal Cells: A Manual of Basic Technique”(2010) :239-260
Thank YouSubmitted by ,
Dr k.Santosh kumar,MVSc veterinary biotechnology,
santoshkmr787@gmail.com