January 15, 2007 (Vol. 27, No. 2)

Point-and-Shoot Technology Makes Isolating Pure Cell Populations Almost a Breeze

Laser capture microdissection (LCM) is emerging as a premier technology with a point-and-shoot capability designed to rapidly isolate single cells or unique multicellular structures from complex heterogeneous tissue. The dissected sample can then undergo a battery of biochemical and molecular analyses that encompass most of the “-omics” such as proteomics, metabolomics, micromics, genomics, etc. Additionally, coupling microarray analyses to the process can provide a more global look at gene expression. Applications for LCM include therapeutic efficacy and toxicity assessments, drug interactions, and even forensics.

The principle and operation of LCM are both simple and elegant. A tissue sample is placed on a glass slide and overlaid with a special thermoplastic transfer film. Like a glue gun capable of pinpoint precision, the laser beam zaps a selected target area bonding it to the film without altering morphology or damaging the sample. The transfer film with the bonded cells is then lifted off the thin tissue section, which leaves all unwanted cells behind. Ejected into a nearby tube, the sample can be automatically harvested for analyses of DNA, RNA, or protein. With the push of a button, this innovative technology can microdissect hundreds of cells within only a few minutes.

Safety and Toxicology Biomarkers

Olivier Grenat, Ph.D., head of the molecular biology group in investigative toxicology at Novartis (www.novartis.com), uses LCM for evaluating safety and toxicity of drugs. “We focused on a specific structure of the kidney, the glomerulus, in order to assess how those cells respond to drug treatment,” said Dr. Grenat. “After capturing those cells, we extracted glomerular RNA and performed gene expression studies. Next, we compared those results to the profile of the whole kidney. These types of studies help to identify safety biomarkers that are relevant to structures such as the glomerulus.”

Dr. Grenat notes that LCM can enrich any area of interest for studies. “We also have microdissected tissue in rat brain to examine the effect on specific areas. We are easily able to capture the effect of compounds here that would be missed if we looked at the entire brain only. This technology allows us to see where a compound goes and how it impacts safety and efficacy in any tissue.”

Several important challenges continue to vex the field, according to Dr. Grenat. “The main obstacle relates to sampling issues. First, RNA can be easily degraded, so the type of tissue and conditions used for capturing and preserving can greatly impact results. Secondly, the amount of sample obtainable is very small. Although use of PCR is helpful, methods for consistent global profiling need to be established.”

Dr. Grenat also sees the field shifting toward the use of more specific markers to identify and enrich single cells of interest. “As more and more specific antibodies become available, we can use these to identify specific structures within tissue bearing biomarkers of interest. For example, we already have good markers to distinguish proliferating cells of the liver. This greatly assists in toxicological analyses.”

LMPC and ES Cells

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of candidates. Researchers at Pfizer (www.pfizer.com) have just validated a novel technique called laser microdissection with pressure catapulting (LMPC) as a means to obtain cardiomyocyte precursor cells from mouse ES cells for in vitro safety pharmacology assays.

“Cardiotoxicity can be a side effect of therapeutics,” say Nestor X. Barrezueta, senior scientist for Pfizer, “It has been a major reason for increased scrutiny toward several drugs being developed and remains a serious concern of regulatory agencies. Cardiomyocytes derived from mouse ES cells provide a model system to better predict cardiotoxicity. However, generation of enriched populations of ES-derived cardiomyocytes is a significant challenge.”

Pfizer scientists employed the technique of pressure catapulting following LCM because it permits precise and noncontact excision of specific cells within a population and prevents contamination from other cells in the culture. A photonic cloud provides an energy pulse and essentially beams the specimen up and into a tube cap.

“This approach is rather unique,” notes Barrezueta, “because while the majority of LMPC applications have focused on mRNA isolation and gene expression analyses in target cells, we showed that it is also a useful tool to isolate differentiated cells from ES cells.”

Is there a wider application for the use of LMPC and ES cells? “Yes, LMPC offers an innovative and quicker alternative to potentially isolate any ES cell-derived cell type. More in-depth investigation is still needed to determine if other such ES cell-derived cell types, such as hepatocytes, pancreatic islet cell precursors, and neuronal precursors, might be manipulated by LMPC for toxicity screenings and drug discovery.”

Contact-free Dissection

As one of the suppliers of LMPC systems, PALM Microlaser Technologies (www.palmmicrolaser.com), a Carl Zeiss subsidiary, offers an LMPC system that uses a contact-free retrieval method.

“Cross-contamination of isolated samples is a big problem with laser microdissection,” said Oliver Prange, Ph.D., application scientist. “We developed a technology to overcome that obstacle by isolating samples in a completely contact-free manner. Basically, what happens is that after laser microdissection, the instrument switches into a collection mode in which a single defocused laser is fired. This, among other effects, causes a gaseous expansion under the excised area that creates a local area of pressure and accelerates the sample against the flow of gravity into a collection vial.”

Adjusting the laser pressure essentially allows someone to capture any specific target without touching surrounding elements. “Some customers are interested in harvesting single cells for microgenomic applications,” says Dr. Prange. “If you were to rely on gravity on this scale, you could have errors in capturing samples. Maybe it won’t fall down due to electrostatics, or it may glide in the air, which is viscous for a single cell.”

Other clients may need much larger amounts of sample for DNA microarray or proteomic applications, for example. “Here the use of automation and robotics is essential,” notes Dr. Prange. “If a scientist wishes to examine only one cell type within a large population, inspection by eye can be unreliable and time consuming. It’s easy when you use integrated automatic hardware and software routines. For example, image analysis, auto focusing, and robotic capturing can be combined into a workflow in which the instrument automatically detects and catapults cells into specific wells on a 96-well plate. Robotics really helps, especially if you have front-end image analysis established.”

Cut and Capture

Molecular Devices (www.moldev.com) provides the Veritas™ Microdissection Instrument, which combines a solid-state UV laser for cutting with an IR laser for LCM.

“This is the first such system on the market,” notes Jan Hughes, vp of worldwide marketing. “Many companies use only a UV laser to cut cells out of a sample. However, we found a sample-dependent problem in this type of laser; it generates heat and can damage RNA. So, we developed instruments that combine both the unprecedented speed and flexibility of a UV laser and the gentle, precise capture of an IR laser. This type of approach does not affect the chemistry or morphology of the cells or tissues that are microdissected.”

At the heart of the system is a thin, thermoplastic film fixed to the bottom of a tiny CapSure® LCM Cap that is placed over the target area. As the laser is pulsed, the film softens, extends down, and adheres to the specific region of interest, as outlined by the user. As the film resolidifies and the cap is lifted away from the slide, so is the sample. Following microdissection, the cap may be coupled to a microcentrifuge tube, in which only a few mL of lysis buffer is needed to extract RNA, DNA, or proteins from the microdissected material.

“One of the major advantages of this system,” Hughes says, “is that you can observe the sample collected before and after the microdissection process. This allows a high level of quality control since you can verify that what you microdissected is what you wanted.”

While the company only offers two LCM instrument systems, it also provides a full solution of microgenomic reagent products that support and complement the LCM technology from staining kits to nucleic acid extraction and RNA amplification kits. “We’re like a one-stop shop,” says Hughes. “The obvious advantage here is that we can offer reagents that have been quality controlled and easily work with our instruments.”

Precise Laser Control

Leica Microsystems (www.leicamicrosystems.com) provides an upright laser microdissection system. Bob Fasulka, director of applications support, says, “Other systems have inverted microscopes, a fixed laser, and moveable stages. The stage is massive. When you have to move the stage, it can cause the precision to decrease over time. We designed an upright microscope in which the laser is guided and the stage is fixed. This allows high precision and fast collection of small objects such as individual cells. The instrument is equipped with up to 150X magnification and can even cut chromosomes.”

Fasulka advises that there are three important strategies for effective use of LCM: eliminate cross-contamination, prevent the laser from touching samples, and quick protection for the excised sample. “The system we use cuts samples on foil slides and allows the dissectate to quickly fall into a collection vial due to gravity. The sample may then undergo RNA isolation or PCR analysis depending on whether you have lysis or reaction buffers in the collection vial. The speed of this reaction better protects such easily degradable products like RNA.”

According to Fasulka, the type of laser used in LCM instrumentation is also important. “We use a crystal diode system that has an excellent beam profile and high-power density. This type of laser allows even thick and hard specimens to be easily cut. Some researchers need to analyze samples from bone, plants, or tissues embedded in plastic, for example. These are traditionally difficult to cut precisely. The Leica LMD6000 has improved optics and exact laser control that allows ease of use when working with such traditionally difficult samples.”

Partnering with Reagent Providers

Molecular Machines and Industries (MMI; www.molecular-machines.com) uses a UV laser to cut around the cell(s) of interest and then harvests the sample with an adhesive cap. According to Ashi Malekafzali, applications manager, “This is an easy and simple technique that enables the performance of any staining protocol necessary without the fear of dehydration or static, as has been seen with other LCM technologies. It is important that the cutting technique has a path as narrow as possible to preserve the sample integrity. While some existing LCM technologies have an average cutting width of five microns, ours consistently allows submicron cuts. Also, having the ability to LCM with the 100X objective enables our users to easily perform LCM on chromosomes and single cells.”

Malekafzali notes that a growing trend in the industry is the formation of partnerships with providers for LCM sample processing. “Many companies, including our own, work with reagent manufacturers to modify their kits to make them more LCM friendly. We then provide the kits along with the instrumentation so that scientists don’t have to reinvent the wheel, in the sense that they can quickly perform their studies knowing that the reagents are compatible with the instrumentation.”

Multiplexing from Sample-limited Specimens

Although LCM can snatch specific cells or tissue from complex specimens, the amount of RNA derived from such capture is miniscule. How do researchers analyze such small samples? Applied Biosystems (www.appliedbiosystems.com) recently launched a new commercial reagent designed to combat this problem.

“Our recently launched TaqMan® PreAmp Master Mix Kit enables accurate, quantitative, and real-time gene expression analysis on small or rare samples,” reports Mark Shannon, Ph.D., staff scientist for the advanced research and technology group. “RNA harvested from the tissue after LCM is reverse transcribed using random primers. The PreAmp Kit performs an intermediate, multiplex amplification step between the reverse transcription reaction and real-time PCR that enriches the cDNA for up to 100 genes, using a pool of TaqMan Gene Expression Assays. This provides a 1,000- to 16,000-fold simultaneous enhancement of the gene targets in the sample. Next, the resulting preamplified reaction is diluted and aliquoted to serve as the starting material for a subsequent singleplex, real-time PCR with each of the individual TaqMan Gene Expression Assays represented in the assay pool.”

According to Dr. Shannon, one of the exciting features of Applied Biosystem’s product is that it enables truly quantitative analysis. “This allows researchers to stretch as little as one nanogram of cDNA into 200 real-time PCR reactions for gene expression analysis for up to 100 gene expression targets with minimal hands-on time.”

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