Cell Culture Automation
Tanuja Koppal

For high-throughput, multi-user laboratories that are involved in running diverse assays and demand high capacity and walk-up capabilities, investing in the right robotic systems becomes critical. There are several factors that come into play when choosing the right system to adopt. Cost and availability of space are always important considerations. Expandable capacity, the ease of use and integration of multiple systems are particularly important for labs looking to grow significantly. Reliability and technical support are critical to labs dealing with multiple users and round-the-clock use. Options for automating are often expensive and labor-intensive and customization is not always feasible. Hence, making the right choices early on is critical. A lot of processes involved in cell culture, which were once performed manually by skilled technicians, are now being automated by robotic systems connected to each other by software programs that help coordinate all the various activities. The use of cells in the drug pipeline has also increased in recent years.

Pharma is now moving towards biopharma, which is driving the need for cell culture automation, says Graham Threadgill, director, Life Science Automation, Discovery Products Business Center, Beckman Coulter Inc. Cells are used for primary and secondary screening in early discovery all the way to drug manufacturing. Hence, the systems designed for automating cell culture are both plate-based and flask-based to accommodate small and large cell volumes. Particularly in drug discovery, more companies are migrating from biochemical to cell-based assays.

There is a continued trend to more cell-based assays and we are observing this trend in the large number of requests that we are receiving for environmentally controlled systems for assays and plate-based cell maintenance, says Debra Toburen, senior product manager, Integrated Systems at Velocity11 (now a part of Agilent Technologies). 50-80% of the assays that some of our customers are running are cell-based.

The robotics for cell culture automation range from the large, motioncontrolled, table-top systems that incorporate several robotic components and can perform multiple washings, incubations, and readings all in one run to those that consist of only the basic components needed for an assay, such as the dispenser, washer and reader. Here, the trend being observed is the replacement of large automation platforms by smaller workstations that are individually managed by a few people, as companies shift their screening strategy from shot-gun approaches with large libraries to screening with smaller and more targeted libraries.

Five years ago we saw giant rooms full of automation and we now find that it is trending down to more individually managed laboratory systems, says David M. Donofrio, Director, Market Development at Molecular Devices (now part of MDS Analytical Technologies). Automation is also becoming less specialized and is being incorporated in various labs within the same organization. In the past, researchers wrote their own methods, programmed and tested it. But now, all they are looking to do is push a few buttons to change a few variables, says Threadgill. You no longer have the automation expert in the organization and so you need to make the automation systems much easier to use, more simple and appliance-like. The automation systems for cell culture are specially designed to offer a protected, contamination-free work area that can be controlled for temperature and humidity. Hence, the software control is turning out to be a critical aspect

For cell culture, seeding and feeding cells can take days if not weeks and the robotic systems are often running overnight. The software has to be able to handle and coordinate all those processes throughout the long time period, says Threadgill. The software also manages data handling and sample tracking to know what is happening to each sample throughout the entire process.

Users are also looking to vendors for more service and technical support and many companies have started offering multiple levels of customer training. We provide on-site, end-user training on how to write a protocol and access the system, says Toburen.

However, for those customers looking to become the resident expert at their company on the use of the platform, they provide more extensive technical training. They [customers] will come on-site and get trained on all instruments and software to help them gain the necessary expertise.

Microarray Technology
Tanuja Koppal

Microarrays spotted with oligonucleotides, DNA, RNA and proteins have been routinely used as tools for expression profiling for more than a decade. What’s exciting about this technology is that it has continually evolved to incorporate new assays, novel probes and diverse design formats to keep up with advances in science and technology. Over the years, microarray technologies have improved to offer better specificity, sensitivity and reliability. However, one limitation inherent to their design has yet to be overcome: the fact that microarrays cannot be used to find something completely novel, since the arrays consist of a set of predefined, pre-spotted genes or proteins. They can never be a truly hypothesis-free discovery platform.

“Microarrays are a starting point and are rarely sufficient for reaching a true biological conclusion,” says Jon Sherlock, product manager of TaqMan Array Plates and Express Plates in Applied Biosystems’ genomic assays business. “Often, you have to take that information to the next level,” says Sherlock, and according to him, the TaqMan arrays aim to do just that. The TaqMan arrays, offered in a card-like format, consist of 384 pre-spotted probes that measure levels of RNA in a sample, using the PCR-based quantitative TaqMan technology. “The TaqMan provides a definitive, quantitative answer and no validation is necessary with any alternative technology,” says Sherlock. This technology is also offered in 96-well plates called TaqMan Express Plates, in which customers can select from more than 50,000 predesigned assays for different targets in rat, human, mouse, dog and other genomes. “[Customers] can select a certain cluster of genes that represent a cellular pathway that they are interested in exploring,” says Sherlock. By May 2009, 130 new assays based on representative gene sets for predesigned pathways—including cytokines; stem cells; kinases; and genes for oncology, inflammation and many others—have been launched on the TaqMan Express Plates. As companies update their arrays with new sets of assays and probes, customers are looking for more cost-effective options that offer higher flexibility and throughput. Agilent Technologies, Inc., recently expanded beyond its comparative genomic hybridization (CGH) arrays into a new area for measuring copy number variation (CNV). “The CNV arrays are an extension of the CGH platform, where we are still looking for DNA copy number changes,” says Dione Bailey, product manager for the CGH/CNV Microarrays at Agilent. The CNV arrays have probes designed for regions of the genome that are fairly complex or are highly repetitive and not unique. “We have now expanded our probe database and catalog offerings to target those known regions of CNV,” says Bailey.

Using Agilent’s eArray web portal, customers can also design their own CGH arrays using information from a database that contains nearly 24 million probes. “Some customers like to design arrays based on their specific needs, while others like to look at our catalog designs and make some minor modifications to them,” says Bailey. “It doesn’t cost you any more to design a custom array than it does to purchase a catalog array. There is no design fee, setup fee or any minimum quantity to place an order.” The flexibility for customers to design their own arrays and choose from different array formats helps reduce experimental costs. Costs can also be reduced by using a technology in which inter- and intra-assay variabilities are low and the amount of hands-on time is decreased. “Customers should be looking for the best value,” says Sherlock. “Having a technology that will work, getting a result that you can trust—without any pre-validation, optimization or post-validation needed, and without any repeats or failures—is very important.”

Microplate Readers
Angelo DePalma

Microplate readers (MPRs) detect light-releasing chemical reactions occurring within the wells of microtiter plates. Since reactions can be associated with biological, physical, or other chemical events, plate readers are popular in the life sciences, particularly in cell biology and drug discovery research.

MPRs may use any one of several types of detection, including absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. Absorbance-based plate readers, which have been around for three decades, operate by measuring the reduction of intensity of light through the plate due to the presence of an absorbing molecule. Luminescence refers to the release of light as the chemical reaction occurs. Fluorescence techniques are all based on release of light at one wavelength as a consequence of excitation at a different wavelength. In fluorescence intensity the excitation and emission are simultaneous; in time-resolved experiments the emitters flash several nanoseconds after the excitation. Fluorescence polarization resembles fluorescence intensity, but includes polarizing light filters along the light path.

Wavelength capabilities, linearity, throughput, sensitivity and read time are the critical features to look for in an MPR. The choice depends on the types of analysis and workload you expect in your lab. Since modern MPRs are relatively similar within an instrument class, the market has become extremely price-sensitive. Prices for readers range from about $6,000 to $20,000. Differentiators include detection modes (more are costlier), scanning with a monochromator or filtering at fixed wavelengths, sample heating/chilling capabilities and software. User-friendliness of software is particularly important in commercial labs where operators are not degreed scientists or when turnover is high. Software that stores and helps generate methods is an important feature for high-throughput labs or those operating in a regulated environment.

MPRs are of two general types: single-mode devices detect one type of signal or wavelength, while multi-mode MPRs detect multiple signals. Single-mode instruments are often dedicated to one type of assay and tend to cost less, while multi-mode devices are more versatile and therefore more expensive.

Less expensive MPRs use single-wavelength filters to dial in the desired wavelength of light whereas more sophisticated instruments employ monochromators, which can be thought of as tunable optical filters. Single-wavelength filters provide high sensitivity but only permit one wavelength through; monochromators can dial in any wavelength. Specialized band-pass filters may be used to refine monochromators further. Monochromators are desirable early in assay development, or when working with unknowns, because they require prior knowledge of assay characteristics.

“Microplate readers have been on the market for a long time,” says Xavier Amouretti, product manager at BioTek Instruments (Winooski, VT). “And nearly every instrument gets decent performance although higher-priced models tend to do better. But the real differentiators are specialized features such as run recognition, software, userfriendliness, and wavelength selection methods.”

BioTek is one of a handful of large manufacturers of MPRs that include PerkinElmer, Molecular Devices (now part of MDS Analytical), Tecan, BMG LabTech, Berthold Technologies, LabSystems (a Thermo Fisher Scientific company), Beckman Coulter, and Turner Bio Systems. Numerous Chinese instrument-makers have sprung up recently in the MPR market, mostly to serve domestic demand. BioTek’s flagship MPR, the Synergy™ 4 Hybrid, combines filter-based and monochromator detection. “A lot of light is lost with monochromators,” Amouretti notes. “Some assays, particularly those with weak signals or employing poorly-fluorescent species, may do better with filters.”,/p>

Modularity, says Barry Landis, Ph.D., detection channel sales manager at Tecan (Research Triangle Park, NC), is a notable trend in how MPRs are designed and ultimately purchased. Landis defines modularity as the ability to mix and match analysis modes as needed. The benefit: users can select and purchase only the functions they desire, and add to them later on without the need to purchase a new instrument. “Until recently, if you didn’t need a particular detection mode—for example absorbance or luminescence—you still had to buy it.” LED (light-emitting diode) excitation and quadruple monochromators represent recent MPR technology advancements. LEDs provide a tenfold increase in the intensity of excitation light compared with conventional excitation mode, xenon bulbs. Provided the sample is not quenched, LED excitation will increase the signal from fluorescence and other assays proportionally to the increase in excitation intensity. The quadruple design uses two monochromators for excitation and two for emission. According to Tecan’s Landis, when used together, these technologies out-perform filter-based systems in terms of sensitivity.

“The extra monochromators block stray light, and with it false peaks,” says Landis.

RNA Technology
Tanuja Koppal

With intense research in such areas as RNA interference (RNAi) and micro RNAs, there is renewed interest in studying and using RNA. DNA/RNA synthesizers are used to synthesize oligonucleotides for a variety of applications that include PCR, sequencing, microarrays, RNAi, antisense and others. Laboratories using RNA on a large scale are looking to synthesize their own oligonucleotides, while others are seeking reliable custom oligonucleotide providers. Reliability, scalability, flexibility, ease of use, throughput, cost efficiency and service are some of the features that people look for.

There are different types of RNA that can be synthesized for different applications—namely, unmodified RNA, modified RNA, RNA conjugates, RNA chimeras and labeled RNA probes. Unmodified RNAs are de-protected, de-salted, endotoxin-free products, while modified RNAs can have a variety of modifications, internally as well as at the 3’ and 5’ positions. Dual-labeled RNA probes have fluorescent quenchers and reporters tagged to the RNA molecule for use in applications such as PCR and microarrays, while RNA conjugates are coupled with cell-penetrating peptides for use in gene expression, antisense, cell delivery and uptake. There are several proprietary synthesis and deprotection technologies that are in use for synthesizing RNAs with high coupling efficiencies, fast de-protection and a high level of purity.

Mark Behlke, MD, Ph.D. chief scientific officer at Integrated DNA Technologies (IDT), deals with multiple scales of RNA synthesis on a routine basis. IDT uses all in-house-designedand- built synthesizers. “We have a column-based platform that handles all medium- to large-scale synthesis and a 96-well plate-based system that does mostly small-scale synthesis,” says Behlke. While the plate-based system handles small-scale synthesis around the 100 nmol range, columns are used for synthesis in the 250 nmol to micromole range and large-scale reaction vessels can be used for synthesis from 10 mg up to 10 g. Synthesizing oligos on a plate-based system is cheaper and more efficient than using the column-based system. However, it’s much more difficult to maintain high coupling efficiency in a plate-based system, because it’s an open architecture system, while a closed column is much easier to keep environmentally sealed from atmospheric water, which is detrimental to nucleic acid synthesis. “Our specially engineered plate-based system can be kept in an isolated and controlled environment that is friendly for oligo synthesis and maintains high coupling efficiency,” says Behlke.

Maintaining high coupling efficiency and product quality is also more challenging with RNA synthesis than it is with DNA synthesis. “Last year we were able to convert the plate-based systems to do RNA synthesis,” says Behlke. Besides synthesis, purification and quality control are also important. IDT has set up an affinity-based purification system to be done in a rapid and cost-effective way on all its samples. “Our base price for siRNA duplexes and dicer substrates is now below $100 for full-catalog single orders, and obviously for large orders, the pricing can be substantially discounted,” says Behlke. “While we can still make HPLC-purified small- and large-scale RNAs like we always have, this approach offers a more economical, highthroughput alternative.”