Characterization of Cell Line


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Characterization is the defining/ outlining those many traits of the cell line………some of which may be unique !

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Characterization of Cell Line

  2. 2. Making Sense!!!
  3. 3. Details not clear??
  4. 4. What about now !!
  5. 5. 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 !
  6. 6. 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)
  7. 7. ♟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
  8. 8. ♫ 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
  9. 9. Criterion/ Method for Characterization… CELL MORPHOLOGY (Freshney, 2010)
  10. 10. 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
  11. 11. CHAPTER I
  12. 12. 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
  13. 13. 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.
  14. 14. ∗ 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
  15. 15. 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
  16. 16. Subconfluent: spatial relationships of the cells are intermediate between sparse and confluent
  17. 17. start to form multilayers Cells that have changed or have been changed in a way that differs to their normal counterparts..
  18. 18. ∗ 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
  19. 19. ∗ Mouse 3T3 cells (Fig. s, t) grow like multipolar fibroblasts at low cell density
  20. 20. ∗ But Mouse 3T3 cells become epithelioid at confluence (Fig. v, w).
  21. 21. ∗ 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
  22. 22. ∗ 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
  23. 23. “When the identity of the cells has not been confirmed, the terms ‘‘fibroblast-like’’ (or ‘‘fibroblastoid’’) and ‘‘epithelium-like’’ (or ‘‘epithelioid’’) should be used”
  24. 24. ∗ Carcinoma-derived cells are often epithelium-like but more variable in morphology (Fig. 15.2m, n, k, l)
  25. 25. ∗ Some mesenchymal cells like CHO-K1 and endothelium can also assume an epithelium-like morphology at confluence (Fig.d, r)
  26. 26. 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
  27. 27. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches
  28. 28. ∗ Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface
  29. 29. 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
  30. 30. 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
  31. 31. ∗ 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”
  32. 32. 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
  33. 33. 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
  34. 34. CHAPTER 2
  35. 35. 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
  36. 36. 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)
  37. 37. ∗ Shows the chromosomes as they appear in metaphase
  38. 38.  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
  39. 39. 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
  40. 40. ♥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
  41. 41. Normal Human Male Karyotype
  42. 42. Normal Human Female Karyotype
  43. 43. Is this Male or Female Karyotype?
  44. 44. Down Syndrome Karyotype ∗ Trisomy 21
  45. 45. Normal Karyotype in Cattle
  46. 46. ♞ 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
  47. 47. 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
  48. 48. ∗ Chromosome analysis can also distinguish between normal and transformed cells because the chromosome number is more stable in normal cells (except in mice)
  49. 49. 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
  50. 50. 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.
  51. 51. 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.
  52. 52. 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
  53. 53. 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.
  54. 54. a) C-banding b) R-banding c) Q-banding d) G-banding
  55. 55. 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
  56. 56. ∗ 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’’)
  57. 57. (A) (C) (B)
  58. 58. 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
  59. 59. ∗ Chromosome paints are available commercially from a number of sources
  60. 60. 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
  61. 61.  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
  62. 62. Spectral karyogram of a human female (Schrock et al., 1996)
  63. 63. 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
  64. 64. 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
  65. 65. 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.
  66. 66. 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
  67. 67. (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)
  68. 68. ∗ 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
  69. 69. CHAPTER 3
  70. 70. It involves three methods: DNA hybridization DNA fingerprinting DNA profiling
  71. 71. 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
  72. 72. 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
  73. 73. ☻ 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
  74. 74. 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)
  75. 75. . D A Gilbert “Application of DNA fingerprints for cell-line individualization”Am J Hum Genet. 1990 September; 47(3): 499–514.
  76. 76. 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.
  77. 77. ∗ 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
  78. 78. 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).
  79. 79. ☻ 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
  80. 80. “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”
  81. 81. REFERENCES D A Gilbert “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
  82. 82. ∗ 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……………
  83. 83. Thank You
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