Cell Culture Harvesting

· Culture types:

· In situ cultures may yield more metaphases than suspension cultures

· Cell culture density:

· Affects spreading

· Removal of mitotic cells (for attached cultures)

· Mechanical

· Rapping flasks sharply on the benchtop

· Scraping with a rubber policeman

· enzymatic

· trypsin

· EDTA

· Mitotic arrest

· Colcemid® (IMPORTANT)

· Mechanism of action:

· Prevents spindle fiber formation

· Sister chromatids are not pulled to opposite poles

· Chromosome condensation

· Concentration and duration alter this effect

· Greater with some cell types:

· Blood, bone marrow, CVS

· Less in others:

· Amniocytes

· Solid tumours

· May be related to cell cycle time

· Effect is mitigated by anticontraction agents

· Ethidium bromide, BrdU, etc.

· Other effects of increased dosage:

· Dramitcally increased mitotic index

· Straightens chromosomes

· Crisps chromatid edges

· Increases chromosome spreading (releases them from mitotic apparatus

· Optimal concentration for each tissue type should be tested for every laboratory

· Concentration may be increased in presence of anticontraction agents

· Colchicine

· Natural version of Colcemid

· Slows cell cycle speed

· May yield longer chromosomes

· May stop division in some cultures

· Velban (vinblastine sulfate)

· Good for tumours and other difficult specimens when Cocemid seems ineffective

· Anticontraction agents:

· Ethidium bromide (EB)

· Causes poor spreading

· Other factors in chromosome contraction:

· Heavy cell growth

· Depleted medium and metabolic byproducts cause irreversible chromosome contraction

· Concentrated cell suspensions during slide making

· Too much debris in cell suspension during slide making

· Hypotonic treatment (MOST IMPORTANT)

· Agents:

· KCl (0.062M)

· Sodium citrate

· Combinations of these

· Affects:

· Spreading

· Note that too high concentrations can cause “imploded” (tight) cells

· Chromatid width and separation

· Elimination of RBCs

· Loss of mitotic cells due to increased fragility during centrifugation

· Chromosomes escaping the cytoplasm

· Mechanism:

· Increase cell volume so that chromosomes can find the space to spread out

· Active process

· If potassium pump is poisoned the cells do not swell

· Modifying factors:

· Temperature of hypotonic solution

· Prewarming to 37^oC may increase effectiveness

· Type of salt

· Sodium citrate yields wider chromatids than KCl

· Ohnuki’s hypotonic yields longer chromatids than KCl

· Cell types:

· Solid tumours, ALL bone marrows

·

· Centrifugation in between steps

· Relative centrifugal force (RCF) (gravities)

· RCF = 0.00001118 x r x N²

· r = rotating radius in cm

· N = rotating speed

· Cells can be lost due to damage from shear forces

· Cells can be lost in the supernatant

· No centrifugation is required for in situ cultures

· Fixation

· “fix” – methanol:acetic acid 3:1

· Note that formalin-fixed cells do not band

· Actions

· Removes water from cells

· Kills and preserves the cells

· Hardens membranes and chromatin

· Preparing chromosomes for the banding procedure

·

· Prefix:

· Makes cells more resistant to damage from centrifugation and the shock of pure fixative

· First fix:

· Drops added first

· Prevents loss of metaphase cells due to breakage

· Temperature:

· Cold fixation may improve chromosome morphology

· Once cells are in first fix they may be left for days or weeks or more before slides are made

· Second and third fixes are just to remove debris and RBCs

· May also help with spreading

· Slide-making:

· Affects spreading

References:

· Barch MJ, Knutsen T, Spurbeck JL. The AGT Cytogenetics Laboratory Manual. 3rd ed. Lippincott Williams & Wilkins; 1997.