Cell Harvest

After the cell cultures have grown for the appropriate period of time and there is a sufficient number of dividing cells, the cells are harvested. Harvest is the procedure of collecting the dividing cells at metaphase, their subsequent hypotonic treatment and fixation, and the placement of the chromosomes on glass slides so they can be stained and microscopically examined. The basic steps of cell harvest are the same for all specimen types, with minor variation. An example is shown in Fig. 1.

Mitotic Inhibitor

A mitotic inhibitor must be used to obtain adequate numbers of cells in the metaphase. Colcemid®, an analog of colchicine, is used in most cytogenetics laboratories. Colcemid binds to the protein tubulin, obstructing formation of the spindle fibers or destroying those already present. This prevents separation of the sister chromatids in anaphase, thus collecting the cells in the metaphase. Exposure time to Colcemid is a trade-off between quantity and quality. A longer exposure results in more metaphases being collected, but they will be shorter because chromosomes condense as they progress through metaphase. Longer chromosomes are generally preferred for cytogenetic studies. Exposure time to colcemid varies by specimen type.

Hypotonic Solution

A hypotonic solution is added to the cells after exposure to Colcemid. The hypotonic solution has a lower salt concentration than the cell cytoplasm, allowing water to move into the cell by osmosis. This swells the cells and is critical for adequate spreading of the chromosomes on the microscope slide. Timing is crucial, as too long an exposure will cause the cells to burst. Too short an exposure to hypotonic solution will not swell the cells sufficiently, which results in poor spreading of the chromosomes.

There are a variety of acceptable hypotonic solutions, including 0.075M potassium chloride (KCl), 0.8% sodium citrate, dilute balanced salt solutions, dilute serum, and mixtures of KCl and sodium citrate. Morphology of the chromosomes is affected by the hypotonic solution used. The choice of hypotonic solution is based on specimen type and laboratory protocol.

Cytogenetics Procedures

slide

CULTURE HARVEST preparation

Fig. 1. Overview of culture and harvest for chromosome analysis. This procedure, with minor variations, is utilized for all specimen types.

slide

CULTURE HARVEST preparation

Fig. 1. Overview of culture and harvest for chromosome analysis. This procedure, with minor variations, is utilized for all specimen types.

Fixative

A solution of three parts absolute methanol to one part glacial acetic acid is used to stop the action of the hypotonic solution and to fix the cells in the swollen state. This fixative also lyses any red blood cells present in the sample. The fixative must be prepared fresh before use because it readily absorbs water from the atmosphere, which adversely affects chromosome quality and staining.

Slide Preparation

The final step of the harvest procedure is slide preparation. Fixed cells from suspension cultures are dropped onto glass slides to allow for subsequent staining and analysis. The concentration of the cell suspension can be adjusted to achieve optimal results. Fixed cells from in situ cultures are not dropped because they are already attached to a cover slip or other solid surface. The prepared slides or cover slips are dried under conditions that favor optimal chromosome spreading and are checked with a phase contrast microscope for metaphase quality and number. A good slide preparation has sufficient numbers of metaphases that are not crowded on the slide, metaphases that are well spread with minimal overlapping of the chromosomes, and no visible cytoplasm.

A number of variables affect the rate of evaporation of fixative from the slide, the spreading of chromosomes, and the overall quality of the slide preparation. Ambient temperature and humidity and length of time in hypotonic treatment all affect spreading of chromosomes. Increased temperature and humidity enhance chromosome spreading, whereas cooler temperature and lower humidity decrease it. Longer exposure to hypotonic solution makes cells more fragile and increases spreading, but an inadequate exposure can result in cells that are difficult to burst. Every technologist must have an arsenal of techniques to effectively deal with these variables.

Other variables in slide making include the height from which the cells are dropped, the use of wet or dry slides, the use of cold, room temperature, or warm slides, the use of steam, air, or flaming the slides, and the angle at which the slide and/or pipet is held.

After slides are prepared they are aged overnight at 60°C or 1 hour at 90°C to enhance chromosome banding. There are also techniques that allow chromosomes to be "aged" by brief exposure to ultraviolet (UV) light.

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