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Characterisation of cell lines

The document outlines the essential aspects of cell line characterization, classifying them into normal and continuous cell lines, and discussing the importance of authentication and provenance. It details the methodologies for analyzing cell morphology and genetic stability, including techniques like karyotyping and DNA profiling. Additionally, it describes various chromosome banding techniques and their applications in studying structural chromosome rearrangements.

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Cell Culture•
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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: ᴥ Simplest and most direct technique to identify cells ᴥ Shortcomings- 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 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 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 species ∗Used to compare the karyotype of one species with the other ∗bands 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 Preparation Duration of the metaphase block may be 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
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.
 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 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 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 number per 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 chromosomes Using 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 hybridization DNA fingerprinting DNA profiling
DNA CONTENT DNA 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 You Submitted by , Dr k.Santosh kumar, MVSc veterinary biotechnology,