Preparing Metaphase Spreads: The Breakdown on Broken-Down Cells

Cytogenetics is a branch of genetics dedicated to the study of chromosomes. Chromosome instability and chromosome rearrangements in the genome are used to diagnose and define several genetic diseases and even some cancers. [1, 2] Researchers use a variety of techniques to identify the structure of chromosomes, including fluorescence in situ hybridization (FISH), karyotyping, flow cytometry, and comparative genomic hybridization (CGH).

Even if you’re not a cytogeneticist, you might need to evaluate the chromosome instability in a certain cell line, for example, before you use them for your thesis project (especially if you are using embryonic stem cells). [3,4]

The first step in assessing chromosome instability or chromosome rearrangement is harvesting chromosomes, which can be a bit of an art form in itself.

First Steps in Preparing Metaphase Spreads

First, your cells need to be about 80% confluent (and healthy!) before you harvest them. If you don’t have enough cells, you may not end up with enough chromosomes for further analysis.

Arresting the Cell Cycle

The next step is to arrest the cell cycle. Arresting the cell cycle in metaphase is crucial for studying chromosomes because this is the point at which they are the most condensed, allowing for easier visualization. A few common metaphase arresting agents include colchicine, demecolcin, vinblastine, and nocodazole. [5,6]

Demecolcin, or colcemid, is the most common compound used to arrest the cell cycle for metaphase spreads. [7,8] Colcemid is a synthetic analog of colchicine, which is highly toxic. [9,10] Vinblastine, like colchicine, is a natural compound found in plants but has also been shown to affect DNA synthesis, which is most likely why it is not used for metaphase spreads. [11,12]

All four of these compounds arrest the cells in metaphase by destabilizing microtubules, which prevents spindle assembly and activates the spindle assembly checkpoint. [5,6] The cells will not be able to proceed into anaphase when incubated with any of these compounds.

Time to Harvest

Once you have arrested your cells in metaphase, it’s time to start harvesting! This almost works the same way as harvesting cell pellets, except you’ll retain the media with the colcemid, rather than pouring it away. Pour the colcemid-spiked media into a conical tube, then trypsinize your cells. Once the cells are detached, dilute the trypsin with the saved colcemid-spiked media. Centrifuge your cells, remove the supernatant, and you’ll have a pellet of metaphase-arrested cells.

Here comes the first tricky part: making the cells fragile. For metaphase spreads, we need to rupture the cell membrane to help visualize the chromosomes.  The cells need to be delicate so that they will burst when dropped onto a microscope slide.

To do this, we need to add a hypotonic solution to the cells, which has a lower concentration of solutes relative to the inside of the cell. This causes an osmotic gradient to form between the outside and the inside of the cells. Thus, adding the hypotonic solution causes the cells to swell, making them very fragile. Think of the cells like water balloons: you want them full enough so that they’ll burst when they hit something, but not full enough to break in your hands!

The easiest way to achieve this balance is to add the hypotonic solution a little at a time, rather than the whole amount at once. This way, you can resuspend your cells slowly, allowing for uniform swelling. You want to make sure that there are no clumps of cells in your tube. Gently tapping on the tube should break up any unwanted cell clumps. The most common hypotonic solution for metaphase spreads is potassium chloride, but some protocols use sodium citrate. [13-15]

Hold That Thought

Once the cells have been incubated with the hypotonic solution, you want to fix them in this swollen state so they can be stored for future use. A combination of methanol and acetic acid seems to be the most common fixation method for chromosome harvesting. Using an alcohol, like methanol, alone will result in the cells shrinking. Adding the acetic acid, which is associated with swelling, preserves the cells in their not-too-swollen state. You can store these cells as a pellet at 4°C for a year or go directly to the next step. [7]

Preparing the Slides

Getting the metaphase chromosome spreads onto the slides is the most difficult part of chromosome harvesting, and there is plenty of debate on which step is the most influential on the final spread.

Once you are ready to make your metaphase spreads, resuspend your cell pellet in fresh fixative a few times. This will help get rid of any cell clumps before you drop them onto your slides.

The trick is to let gravity do the work for you, but you may need to spend some time optimizing the distance. If the distance between your slide and your pipette is too large, your chromosomes will spread too far apart. When chromosomes are too far apart, it may be difficult to determine if they were from one cell or two cells. This could be a huge problem if you are trying to assess the ploidy of your cells. [16]

If the distance between your slide and your pipette is too small, your chromosomes will overlap and be squished together. When your chromosomes are overlapping, you won’t be able to tell them apart. This will make any staining you might do difficult to evaluate. Use a disposable transfer pipette to drop your cells onto clean slides, but keep in mind that the diameter of the pipette can also influence chromosome spread on the slide.

Humidity is a critical component of the metaphase spread procedure, and it seems to be the most important factor affecting how the chromosomes spread on the slides. [17] The fixative pulls moisture from the air, which results in rehydration of the now broken cells. Controlling this rehydration process is the key to beautiful metaphase spreads!

The two most common methods of maintaining slide humidity are:

• keeping the slides slightly damp with water (either by dipping the slides in water or pre-freezing them) before dropping the cells onto them, then allowing them to dry on a heat block.
• using dry slides, dropping the cells, and then immediately placing the slides on a tray in a covered water bath.

Choosing the method is up to the individual, but the overall message is to keep it humid!

When drying the slides, the temperature can vary from 20°C to 75°C depending on the protocol. [6,7,13,18,19] A higher temperature can cause the fixative to dry too quickly. A temperature that is too cold could cause an increase in moisture on your slides, as cold air can’t hold as much water as warm air. This could cause your fixative to dry too slowly. As drying time influences chromosome spreading, it is important to find the right combination of temperature and humidity to optimize chromosome spreading for your cells. [17]

Final Thoughts on Preparing Metaphase Spreads

Once your slides are dry and the chromosomes are fixed in place, they are ready for any downstream applications such as immunostaining. It might be helpful to check your first slide before preparing the next one. That way, you can make any adjustments to your methods (such as distance, humidity, and temperature) based on the chromosome spreading you see under the microscope.

Above all, keep in mind that perfecting your metaphase spreads is an art! Following a protocol step-by-step is a great place to start, but you’ll need to optimize your methods for your needs. If you need a visual protocol or some troubleshooting tips, check out Chromosome Preparation from Cultured Cells by Howe et al. [5]

Do you have any tips for troubleshooting your chromosome harvests? Let us know in the comments.

References:

1. Wang, N., “Methodologies in cancer cytogenetics and molecular cytogenetics”, American Journal of Medical Genetics vol. 115(3), p118–124, 2002.
2. Hochstenbach, R., et al., “A survey of undetected, clinically relevant chromosome abnormalities when replacing postnatal karyotyping by Whole Genome Sequencing”, European Journal of Medical Genetics vol.62(9), 103543, 2019.
3. Bolhaqueiro, A., et al., “Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids”, Nature Genetics vol.51, 824–834, 2019.
4. Miura, M., et al., “Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation” Stem Cells vol.24(4), 1095-1103, 2006.
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12. Williams, J., Carpentieri, U.” Metaphase Arresting Compounds in Embryos”. Nature vol. 216, 613–614, 1967.
13. Michelland, S., et al., “A reliable protocol for high-quality metaphase spreads for in situ hybridization”. Technical Tips Online vol. 3, 126-127, 1998.
14. Kotsarenko, K., et al., “Karyotype changes in long-term cultured tick cell lines”. Sci Rep vol.10, 13443. 2020.
15. Utsumi, K., et al., “Studies on the Structure of Chromosomes I. The Uncoiling of Chromosomes Revealed by Treatment with Hypotonic Solution: Cell Structure and Function vol. 1, 93-99, 1975.
16. Bakker, Bjorn et al. “How to count chromosomes in a cell: An overview of current and novel technologies.” BioEssays: news and reviews in molecular, cellular and developmental biology vol. 37,5, 570-577, 2015
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