Isolating individual cells for molecular analysis

High-throughput technology is continually being invented and refined to allow increasingly sensitive molecular analysis of tissue samples. As new cutting-edge microanalytical procedures and instruments are developed, more rigorous demands will be placed on sample preparation techniques. Using laser-capture microdissection (LCM) in combination with these sensitive analytical methods, researchers can obtain more accurate data and address previously unanswerable questions.

The power of proteomic and genomic techniques relies heavily on the preparation of homogeneous cell populations. Although flow cytometry has long been used to enrich a particular cell type in suspension, the ability to purify cells from solid tissue samples, such as biopsies, has lagged. Another disadvantage of flow cytometry is the requirement of a specific marker for selection. To improve cell isolation, several microdissection methods have been developed, but these are often time-consuming and imprecise.

As an alternative, Michael Emmert-Buck and colleagues at the National Institutes of Health (NIH) in Bethesda, MD, developed LCM and demonstrated its usefulness for studying various tissue types at the DNA, mRNA, and protein levels. The NIH group eventually joined forces with Arcturus Engineering, Inc. (Mountain View, CA; www.arctur.com), as part of a Collaborative Research and Development Agreement for the commercialization of this technology. In addition to its elegant simplicity, LCM has the advantage of preserving the tissue’s original morphology while avoiding contamination from surrounding tissue. Samuel Chu, technical support specialist at Arcturus, explains, “The benefits of microdissection include the ability to procure a pure population of cells that would give y

Figure 1. Laser-capture microdissection. Individual cells are pulled from a tissue sample by directing the laser through the transfer film over a selected cell (1). As other cells are desired, the glass slide can be moved and the laser targeted to hit the new cells (2, 3). The laser attaches the cells to the transfer film, and they are simply lifted from the main tissue for further analysis (4).

ou the most accurate and representative data and results.”

What is LCM?
The LCM system developed at NIH and Arcturus is basically an inverted microscope fitted with a low-power near-infrared laser. Tissue sections are mounted on standard glass slides, and a transparent, 100-mm-thick, ethylene–vinyl acetate film is then placed over the dry section (see Figure 1). The laser provides enough energy to transiently melt this thermoplastic film in a precise location, binding it to the targeted cells. The laser diameter can be adjusted from 7.5 to 30µm so that individual cells or a cluster of cells can be selected. Because the plastic film absorbs most of the thermal energy and the pulse lasts for a fraction of a second, little or no detectable damage of biological macromolecules occurs.

After the appropriate cells have been selected, the film and adherent cells are removed, and the unselected tissue remains in contact with the glass slide. (For QuickTime movies of this process, visit www.arctur.com/lcm_movies.html.) These cells can then be subjected to appropriate extraction conditions for ensuing molecular analysis. To improve the convenience of the technique, the transfer film can be mounted on a transparent cap that fits on a 500-µL microcentrifuge tube.

LCM is compatible with various common methods for the preparation of tissue sections. Tissues are typically fixed by alcohol-based precipitation techniques. Aldehyde-based fixation may also be used, but covalent cross-linking of macromolecules can potentially interfere with subsequent analysis of RNA or protein. Sections of 6-mm thickness can then be prepared from paraffin-embedded or frozen tissue and mounted on glass slides. These sections may be stained by standard techniques such as hematoxylin and eosin, methylene green nuclear stain, fluorescence in situ hybridization, or immunohistochemistry for identification of tissue morphology and cell populations of interest. Because the section thickness is less than that of a cell, up to 20 cells may need to be selected to obtain a complete genome or expression profile. Up to 3000 transfers can be performed on one film, representing more than 6000 cells, depending on their size. The cells are then lysed and extracted in an appropriate buffer for the analysis of DNA, RNA, or protein (see Figure 2). Remarkably, single cells captured by this technique have been successfully analyzed by techniques based on nucleic acid amplification.

The major disadvantage of LCM is that it isolates minute amounts of material, which limits analysis to amplification-based techniques or collection of numerous cells, as in protein analysis. Although this technique is faster than previous microdissection methods, isolation of large numbers of cells from many sections can require considerable time. Another disadvantage is that cover slips and mounting solutions are not compatible with LCM a

Figure 2. Wash and set. The cap to which the cells are attached is placed on a 500-mL tube containing an analysis or wash buffer. The cells are then removed from the cap and further processed for analysis by techniques such as 2D-PAGE, microarrays, and sequencing.

nd, as with other microdissection techniques, visualization of samples can be difficult. Arcturus has equipped its microscopes with a diffusion filter to improve the viewing of cells. For an experienced histopathologist, this suboptimal visualization should not be a problem. Another limitation of LCM is that it is not compatible with live-cell analysis, but for these applications flow cytometry may be used.

Post-LCM applications
Cells isolated from complex tissue by LCM have been successfully studied by various analytical techniques. One of the most prominent methods for examination of microdissected tissue, cDNA microarrays, has been used to compare gene expression profiles between various cell types within a tissue. This has been particularly advantageous in identifying the differences between expression levels in normal and diseased tissues. Alternatively, serial analysis of gene expression has been used to monitor mRNA profiles. Beyond simple mRNA measurements, cDNA libraries have been generated from LCM-purified cells. Analysis of DNA from microdissected cells has included polymerase chain reaction (PCR) amplification of alleles to observe the loss of heterozygosity and detection of mutations by single-strand conformation polymorphism. Quantitative, real-time PCR instruments are also likely to be used successfully with LCM-prepared samples.

Complementary to these DNA and RNA analyses is the examination of differential protein-expression profiles by SDS-PAGE and 2D-PAGE. Mass spectrometric sequencing, peptide mass fingerprinting, in-gel zymography, or Western blot has then been used to identify proteins of interest. Newly developed protein chips may also prove useful to this end. Protein analysis of microdissected tissue can provide important information not accessible with nucleic acid-based techniques, including protein stability and translation efficiency, post-translational modification, protein–protein interaction, and protein–DNA interaction.

Combining LCM with several of the genomic and proteomic techniques mentioned above, the Cancer Genome Anatomy Project (www.ncbi.nlm.nih.gov/CGAP) at the NIH has established an effort to document the progression of normal cells to premalignant and metastatic cancer cells in various tissues. This NIH-based initiative is taking a five-sided approach to this problem. These approaches will use cDNA microarrays to identify genes expressed during mouse and human tumor development, 2D-PAGE and mass spectrometry to profile molecular differences between normal and diseased tissue, and PCR-based techniques to identify genetic polymorphisms and chromosomal aberrations.

Besides their application to disease research, microdissected sections from genetic model organisms such as flies, worms, zebrafish, and mice may lead to new insights in developmental biology. Comparison of gene expression patterns in neighboring cells can be analyzed to improve our understanding of cell fate and cell differentiation. Similarly, differential expression between cell types in particular tissues has already been examined, as in the case of adrenomedullary and adrenocortical cells. “LCM is still in its infancy, and new applications are continuing to be explored,” Chu states.

Conclusions
LCM is challenging analytical techniques to become more sensitive and has already been shown to improve the quality of results obtained by existing molecular methods. “As LCM is helping to push the limits of sensitivity, accurate and reliable analytical techniques will need to be able to meet the demands placed on them,” Chu forecasts. It is clear that in the analysis of pure cell populations, sequence and expression databases will continue to be invaluable tools leading to the identification of novel genes, their functions, and their potential as pharmacological targets. The era of proteomics, microarrays, and bioinformatics is upon us. With the aid of more sensitive analytical techniques, LCM instruments are sure to become common fixtures of many biomedical research facilities.

Suggested reading

1. Emmert-Buck, M. R. et al. Laser capture microdissection. Science 1996, 274, 998–1001.
2. Bonner, R. F. et al. Laser capture microdissection: Molecular analysis of tissue. Science 1997, 278, 1481–1483.
3. Simone, N. L. et al. Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends In Genetics 1998, 14, 272–276.
4. Banks, R. E. et al. The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis: Preliminary findings. Electrophoresis 1999, 20, 689–700.
5. Curran, S. et al. Laser capture microscopy. Mol. Pathol. 2000, 53, 64–68.

Ed Brignole is a freelance writer and researcher at Johns Hopkins University in Baltimore, MD. Comments and questions for the authors may be addressed to the Editorial Office by e-mail at mdd@acs.org, by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.