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