Week 5 2024 Tissue Culture and Cytogenetic Methods PDF

TISSUE CULTURE AND CYTOGENETIC METHODS TISSUE CULTURING AND HARVESTING CLINICAL SPECIMEN TYPES AND HARVESTING PROCEDURES Specimen Types  Amniotic Fluid  Chorionic Villus Sample – CVS  Products of Conception - POC  Skin Biopsy  Peripheral Blood  Tumour Biopsy  Bone Marrow Constitutional Acquir...

TISSUE CULTURE AND CYTOGENETIC METHODS TISSUE CULTURING AND HARVESTING CLINICAL SPECIMEN TYPES AND HARVESTING PROCEDURES Specimen Types  Amniotic Fluid  Chorionic Villus Sample – CVS  Products of Conception - POC  Skin Biopsy  Peripheral Blood  Tumour Biopsy  Bone Marrow Constitutional Acquired TISSUE CULTURE REQUIREMENTS  Suitable culture conditions  Suitable nutrients  Maximise numbers of cells in division  Determination  Method of the optimum time to harvest of capturing the cells in division CULTURE REGIME  Substrate Culture: amniotic fluid, CVS, POC, skin and tumour biopsy  Suspension culture: Blood, Bone Marrow and Lymph Node SUITABLE CULTURE CONDITIONS 5% CO2 Incubator operating at 37ºC Tissue culture vessels to optimise cell adherence STERILE conditions SUITABLE NUTRIENTS  Tissue culture media (Basal media and supplements) Fetal Calf (or Bovine) Serum  Amino acids (L-glutamine)   Growth enhancers  Antibiotics  Antifungals MAXIMISE NUMBERS OF DIVIDING CELLS Blood cultures: Mitogens are used to promote cell division for constitutional analysis - Phytohaemagglutinin (PHA ) Other tissue types have spontaneously dividing cells Some adverse clinical conditions may not exhibit spontaneously dividing cells – CLL, MM and some Lymphoma's OPTIMUM HARVEST TIME      The best time to harvest is when the cells are in there most active phase Most human tissue have a cell cycle time of 16-20 hours Bloods (for constitutional studies) are usually in culture for three days (72hour cultures) Bone marrows and leukaemic bloods are harvested after 1 to 4 days Tissues grown on a substrate may take 7 – 30 days to produce enough cells (Prenatal tissues will usually stop growing after ~50 divisions) CELL CYCLE    Understanding the cell cycle is fundamental to good harvesting Cells should be ACTIVELY dividing Optimisation of cell activity is necessary CAPTURING CELLS IN DIVISION If not in division then chromosomes are not visible  By preventing spindle formation, chromosomes cannot travel to the poles; the cell cannot continue to divide  Colchicine/Colcemid® is a chemical that interferes with spindle formation  Determining best harvest time o Tissue (substrate) cultures can be directly observed with an inverted phase microscope o Suspension cultures cannot be directly observed so “calculated guesses” are applied o Maximum cell yield from actively dividing tissue, resulting in a high mitotic index is the aim of any harvest MITOSIS BLOOD AND BONE MARROW CULTURES SYNCHRONISATION    Primarily used in blood and bone marrow cultures, it is a technique developed to maximise the number of dividing cells at the point of harvest Initially cells are stopped or 'blocked' at S or G2  An example blocking reagent would be Thymidine The cells in culture are held at this point over a period of time to maximise the number of dividing cells present SYNCHRONISATION CONT.  After a set exposure time, the effect of the ‘blocking’ agent will be removed – referred to as ‘release’  To counteract blocking agent Thymidine: Deoxycytodine (DCT) can be used  Cells will the resume the cell cycle and proceed to cell division – MITOSIS  Once the cells enter division, Colcemid® will be added to the cultures to halt the cells at prometaphase SYNCHRONISATION CONT. Mitotic arrest at prometaphase i.e. Colcemid Mitosis P M A T Gap 2 Gap 1 Releasing agent i.e. deoxycytidine S phase Blocking agent i.e. thymidine HARVESTING CELLS  Involves the collection of cultured cells to allow chromosomes to be analysed  Cells are washed to remove the culture medium  They are then subjected to a hypotonic solution to swell the cells  Cells are washed to remove excess cytoplasm. For blood and bone marrow cultures, removal of red blood cells is also required  Finally cell suspensions are “fixed” so that they can be spread onto glass slides and stained for analysis BLOOD HARVEST PROTOCOL  Culture Blood cells are cultured in suspension in growth media with the addition of phytohaemaglutanin (PHA) to stimulate the lymphocytes to divide.  Cultured for 48-96hrs  Synchronisation and release  Colcemid® addition   Hypotonic    Fixative   Made from 3:1- methanol: acetic acid mix to harden and fix the chromosomes (Carnoy’s fixative) Slide preparation   The cells are exposed to a hypotonic solution (eg KCl) The addition of diluted acetic acid (to remove the remainder of the RBC and remove cytoplasm from lymphocytes) Cell suspension is “dropped” on to microscope slides Banding and Staining ready for analysis PERIPHERAL BLOOD HARVEST 1 2 3 4 Aging + G-banding 1. Whole blood in culture media (RPMI, +FBS, +L-glut, +PHA, +pen/strep) Synchronisation complete 2. Addition of hypotonic KCl Lysis of red blood cells and swelling of white blood cells (lymphocytes) 3. Prefixation: addition of 4% Acetic acid Further break down remaining red cells Start to harden chromosomes 4. 3:1 Methanol:Acetic Acid fix washes Harden and preserve chromosomes in preparation for slide dropping. BLOOD HARVEST TROUBLE SHOOTING Problem/Observation Possible Solutions Lots of interphase cells but no metaphase cells No mitogen added to culture: hence no dividing cells Metaphase cells appear squashed and tangled Lots of partial metaphases and random chromosomes in preparation: “chromosome soup” No cells or broken cells Lots of interphase cells but very low mitotic index Hypotonic solution is at the wrong concentration (too salty), or Has not had enough incubation time to swell the cells Hypotonic solution is too weak: cells have swollen and exploded, or cells were treated too roughly during the harvest procedure. Have removed pellet with supernatant, or Possible microbial contamination. Synchronisation step not conducted properly (too early or reagent missed) BLOOD “DROPPING” TROUBLE SHOOTING Problem/Observation Possible Reason Possible Solution Scattered chromosomes Humidity too high Small encased metaphases Humidity to low Hypotonic solution incorrect Clumped cells and metaphases Preparation too sparse or dense Suspension not resuspended correctly Fixative washes not sufficient Cell concentration is incorrect Use air conditioner to reduce humidity and increase temperature Drop preparation closer to the slide Increase humidity and lower temperature Increase the height of the drop Add a fixative “chaser” drop to the preparation Drop onto a wet slide Make sure suspension has been fully re-suspended (do not be too rough this will shatter the cells) Add additional fixative resuspend pellet and centrifuge again Add additional fix if too dense to dilute prep Re- centrifuge sample and remove extra supernatant PRENATAL TISSUE CULTURE  Amniocentesis        A needle is placed through the uterine wall into the amniotic cavity and amniotic fluid is aspirated under ultrasound guidance Usually performed from 15-18wks gestation Cells are concentrated by centrifugation and are inoculated into flasks or onto coverslips contained within petri dishes (in situ cultures) A recommendation of a minimum of three cultures should be created and they must be grown in two independent incubators and two different lots of media are to be used Cells will settle down and grow directly onto the slide/flask surface forming colonies Cells are assessed regularly to determine rate of growth and when they will be ready for harvest. Cells are harvested and fixed onto the coverslips on which they were originally growing ‘in situ’ COLONY FORMATION Fibroblast images from AGT Manual PRENATAL TISSUE CULTURE  Chorionic villus sample (CVS)        A small sample of placental tissue is aspirated either trans-cervical or trans-abdominal with an 18-20 gauge needle. The needle does not pass through the amnion Usually performed from11-14wks gestation Villi must be cleaned to remove traces of blood and maternal decidua before it can be cultured Villi is treated with an enzyme (trypsin or collagenase) to break down the cellular matrix and expose the mesenchymal chore of the villi Cells are inoculated into flasks- minimum of two independent cultures; separate incubators; different media lot# Cultures assessed daily for growth, when adequately grown cells are lifted from the flask surface and are inoculated onto coverslips. Coverslips are then harvested, banded, stained and analysed THE PLACENTA Color Atlas of Fetal and Neonatal Histology pp 363-388 FIBROBLAST TISSUE CULTURE AMNIO OR CHORIONIC VILLI Amniotic fluid is more reflective of the true fetal karyotype where as villi is from a more distantly related cell line and be more likely to affected by confined placental mosiacism  Amniotic fluid is less likely to show maternal cell contamination  CVS can be taken at 11wks gestation. Amniocentesis can not be performed until 15wks.  Recent studies have determined there is no increased risk of fetal loss with invasive procedures (PMID: 26581188, PMID:25042845)  EMBRYONIC CELL DIFFERENTIATION image from: http://www.ufrgs.br/imunovet/molecular_immunology/development.htm BONE MARROW A needle is inserted into the bone marrow and a liquid bone marrow sample is aspirated.  A bone marrow trephine may also be collected which will often be used to provide a morphological examination of the bone marrow structure and cellular make up.   Before culture, trephine samples must be macerated to release the cells from the bony structure BONE MARROW  Bone marrow suspension cultures will have different culturing protocols (synchronisation/mitogens) depending on the cell lineage of the suspected leukaemia     Cells of interest are generally spontaneously dividing and will not require the addition of a mitogen. However some leukaemic conditions generally do not produce useable metaphases and so molecular methods are employed to look for the most clinically relevant chromosomal abnormalities   Myeloid Lymphoid For example TP53 gene deletion in CLL patients. Cell selections: cell populations can be isolated to increase the yield of relevant cells for culture or DNA extraction.  In plasma cell conditions, such as Multiple Myeloma, the surface antigen CD138 produced by mature plasma cells, is used to isolate these cells using antibody complexes with attached magnetic particles. The cells can then be run through a magnetic column to separate the CD138+ cells from the remaining cells. CHROMOSOME BANDING BANDING METHODS The banding techniques fall into two principal groups: 1) those resulting in bands distributed along the length of the whole chromosome,  such as G-, Q- and R-bands 2) those that stain a restricted number of specific bands or structures. C bands centromeric bands  nucleolus organizer regions, NOR's (at terminal regions of active acrocentric chromosomes).  SOLID STAINING o o o This results in a uniform, unbanded appearance Largely obsolete However can be useful for studies on chromosome breakage G BANDING  Generally the preferred method for the routine staining of chromosomes     Simple Slides can be kept for a long time without deterioration General method to obtain G-banding involves treating the slides with a protease such as trypsin, older methods include incubating the slides in hot salinecitrate solution Banding pattern obtained are thought to reflect both structural and functional composition of the chromosome G-BANDING  G-Dark bands      Replicate mid to late in S phase Condense early in mitosis Gene poor tissue specific genes A-T rich DNA Enriched in LINES (long interspersed elements)  G-Light bands      Replicate early in S phase Condense later in mitosis Gene rich housekeeping genes C-G rich DNA Enriched in SINES (short interspersed elements) G BANDS: METHOD Trypsin G-banding 1. Incubate the slides for approx. 10-40 secs in trypsin solution 2. Rinse slides thoroughly with PBS/Gurr buffer 3. Stain in Leishmann/Geimsa solution 4. Rinse slides in distilled water and air-dry 5. Coverslip G-BANDING TROUBLE SHOOTING Problem/Observation Reason Possible solution Fuzzy grey/grainy chromosomes with poor banding definition Slides not aged enough Age slides for longer by leaving an extra day before banding or heating on hotplate / in oven Fat, pale, fuzzy chromosomes Over trypsinised Decrease trypsin time Over all dark chromosomes lacking definition between bands Under trypsinised Increase trypsin time Pale chromosomes but good banding definition Not enough stain Increase stain time/ concentration Dark chromosomes but good banding definition Too much stain Lower stain time/ concentration Smeared and scraped chromosomes Wiped the wrong side of the slide Re-drop slide Q BANDING Quinacrine dihydrochloride  Is an acridine dye that binds to DNA either by intercalation or by external ionic binding  First banding method used on human chromosomes  Roughly correspond to G bands  Limited use due to fluorescence image analysis C-BANDING (CONSTITUTIVE HETEROCHROMATIN) CH is composed of simple sequence, highly repetitive DNA  Darkly staining C-bands are located at the centromeres of the chromosomes, Yq, 1q, 9q and 16q  Marked polymorphism is present in the size of the Cbands, relatively stable, inherited  Replicates late in S phase, lacks active genes  It is used in the investigation of chromosome rearrangement near centromeres and investigating polymorphism  C-Band Male C-banded karyotype REVERSE BANDING      Bands that appear pale by Gbanding, stain darkly by Rbanding Good tool for the detection of abnormalities involving the ends of chromosomes R-banding may be produced by incubation in a very hot saline solution then staining The chemical basis for the staining reactions remain obscure Contrast between light and dark bands is often insufficient NUCLEOLAR ORGANISER REGION (NOR) STAINING   NOR’s contain the genes for 18S and 28S rRNA on chromosomes 13, 14, 15, 21, 22 Regions in which these genes are thought to be actively transcribed can be selectively stained using silver nitrate     NOT all acrocentrics will stain The stain is thought to identify selectively a protein adjacent to the NOR rather then the NOR themselves Chromosomes form different individuals will stain more of less intensely in a consistent manner Therefore a specific staining pattern will be seen for each individual and this polymorphism is a heritable characteristic NOR Visible are eight of the ten acrocentric chromosomes with their stalk regions staining darkly. NOR 46,XY,t(9;15) DIFFERENTIAL REPLICATION STAINING    Different parts of chromosomes replicate at different times BrdU is a thymidine analogue that is readily incorporated into chromosomes When BrdU-substituted chromosomes are subsequently stained with bisbenzimidazole dye Hoechst 33258, a compound that binds to DNA at A+T base pairs, there is quenching of the bright fluorescence normally seen when unsubstituted chromosomes are stained with this dye DIFFERENTIAL REPLICATION STAINING  If BrdU is added to the last part of one cell cycle (B-pulse)   If BrdU is added early (T-pulse)    Chromosome regions replicating EARLY will stain brightly will resemble R bands Chromosome regions replicating LATE will stain brightly and will resemble G bands Homologues usually have similar banding patterns, except of the X chromosome If BrdU is present for 2 full cycles, one chromatid contains DNA with BrdU substituted into one polynucleotide chain while the sister will contain BrdU substituted into both chains:  sister chromatid exchange BLOOMS SYNDROME Sister Chromatid Exchange (left is normal; right is Bloom Syndrome) TELOMERE BANDING    The terminal bands or telomeric regions can be selectively stained by T-banding It is specialised case of R-banding, in which a more destructive treatment results in diminished staining except at terminal bands Telomeres are more effectively visualised using FISH with terminal repeat or sub-telomeric repeats sequence probes BANDING APPLICATIONS o Heterochromatic variants o o o Centromeric variants o o o Large heterochromatin variant, especially 15 & 22. These will stain strongly with C-banding Satellites on chromosome other then acrocentrics o o o Should be C-banded Large short arm variants of acrocentrics o o Chromosomes 1,9 or 16 C banding Large centromeric regions in other chromosomes o o To differentiate between normal variation of chromosomes and structural rearrangements producing genetic abnormality C band This results forma structural rearrangement involving an acrocentric chromosome and cannot be considered a normal variant C-banded and NOR banded Y chromosome variation o o o This is common C-banding, Q banding or FISH can identify an extra material as heterochromatin Small Y, best followed up by looking at the paternal Y THREE-LETTER CODE: GTG, GTL   The Paris conference (1971) introduced a three-letter code to describe the various banding techniques First letter   Second letter   Denotes the general technique (H,F,T,B) Third letter   Denoted the type of banding (G,Q,R,C,T) Denotes the stain used (G,L,A,Q) Not all fall into this classification; NOR MOLECULAR METHODOLOGY o FISH probe for specific parts of the genome from whole chromosomes to microdeletion sites. o QFPCR (quantitative fluorescent PCR) – rapid prenatal aneuploidy diagnosis o MLPA (multiplex ligation dependant probe amplification) o CGH (comparative genomic hybridisation) o aCGH or Chromosome Microarray Methods for detecting DNA copy number changes Mbp Cytogenetic analysis (Karyotype) FISH (metaphase, interphase) PCR (multiplex QF-PCR, real-time PCR) MLPA DNA microarrays, Array CGH Next Generation Sequencing (NGS) Sanger sequencing bp Move towards the molecular karyotype Conventional Cytogenetics: Whole genome analysis at a resolution of 3-5 Mb FISH, QF- PCR, MLPA: Higher resolution but limited to known loci Microarrays: Whole genome analysis of copy number changes. Thousands loci tested in single experiment. Potential for high resolution. 1 Mb down to 20 kb. FLUORESCENCE IN SITU HYBRIDISATION FISH 1-5 Kbp within a 1-2 Mbp chromosome band Fluorescence: the material fluoresces on exposure to a beam of light, emitting a specific wavelength of colour in situ: in place, in this case, on the slide in the metaphase or interphase cell Hybridisation: to join together Is a method using DNA probes to bind to specific sequences on chromosomes in cytogenetic preps (interphase or metaphase) and detecting these probes by labeling them with fluorescent dyes METHOD Specific DNA sequences isolated to make a DNA or RNA probe (200-500 bp) Reporter molecule attached – biotin, digoxigenin Fluorescent label is attached – direct, indirect Labelled sequence hybridised back onto the target DNA Post hybridisation wash to remove mismatched or unhybridised probe – salt conc. (stringency), time, temp Counterstain – DAPI (4,6-diamidino-2-phenylindole) Microscopy and Photography FISH METHOD (QUICK METHOD) Pre-treatment (optional, but creates better signals) Age slides Pepsin pre-treatment MgCl2 /37% formaldehyde Dehydration (EtOH wash series) Denaturation and hybridisation Add probe, coverslip then seal with rubber cement (the probe already has its fluorescent label attached) Denature for 3-5mins at 73°C Hybridise by incubating overnight at 37°C Stringency washes 2xSSC/NP40 wash 73°C 0.4xSSC/NP40 wash at RT Immunodetection Not required for most commercial probes they come pre-labelled for laboratory convenience Counterstain and coverslip DAPI (4,6-diamidino-2-phenylindole) Fluorescence microscopy  Karyotype - Analysis of G-banded chromosomes 3-5 Mbp  FISH – 100bp-5 Kbp within a 1-2 Mbp chromosome band FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy is required for visualization of the bound probe signals TYPES OF FISH PROBES Repetitive sequence probes Centromeric or alpha satellite DNA probes centromeres of each chromosome can be distinguished with individual probe – Except 13 & 21 and 14 & 22 – Used for identifying – aneuploidy , mosaicism, markers – Whole Chromosome Paints Designed to mark the entire chromosome of interest Useful for identifying marker chromosomes and complex rearrangements eg insertions – Small rearrangements of