For several types of drug screening applications, cell-based assays are more relevant than biochemical assays, and in some cases essential. However, many pharmaceutical and biotech companies are still sometimes hesitant to incorporate them into screening programs because of the difficulty and cost of using appropriate cell lines, according to presentations at Cambridge Healthtech Institute’s 11th Annual Cell-Based Assays for High-Throughput Screening conference, held May 17-18 in Philadelphia.
The cell-based assay track was part of CHI’s World Pharmaceutical Conference, and coincided with sessions focused on topics such as ADME-Toxicity, cheminformatics, hit-to-lead, and R&D strategies.
Like its sister tracks, the cell-based assay track drew a mix of representatives from pharmaceutical, biotech, and self-styled “drug discovery” companies, as well as from companies offering bioassay services and products. According to CHI, over 700 people attended at least one of the application-specific tracks. More than 50 percent of the attendees represented pharma, and more than 30 percent represented biotech, CHI said.
The conference, which is one of only a few focused purely on cell-based assays, served primarily “as a forum for pharmaceutical companies, biotechnology companies, and vendors to exchange ideas about cell-based assays,” said Guido Zaman, section head for lead discovery at NV Organon, and one of the conference’s approximately two dozen speakers.
In addition, more than 40 companies exhibited at the conference, and a similar number of individuals or groups presented scientific posters. Paul Wylie, a senior scientist at TTP Labtech of Royston, UK, won the cell-based screening poster competition with a presentation of his screening method for members of the MAP kinase family of proteins using TTP’s Acumen Explorer laser scanning cytometer (see inside story in this issue: TTP Instrument Finds Niche ...).
The Pharma Perspective
The first day of the conference consisted of researchers from various pharma and drug discovery companies sharing their experiences and advances with cell-based assays, as well as discussing the myriad problems and roadblocks that still exist.
Most described applications for which cell-based screening was absolutely essential. For example, Lisa Minor, principal scientist at Johnson and Johnson, discussed using biochemical versus cell-based screens for tyrosine kinase receptors.
“A cell-based assay is the only way you’re going to detect receptor activators,” she said. “You really have no choice.” One of the major advantages of this approach is that one can subsequently measure downstream functionality associated with receptor activation.
She added that there are several tools with which to conduct such cell-based assays, including Western blot — which is “definitely not high-throughput,” she said — Luminex bead-based assays, ELISA, DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay), and the “captured enzyme” method.
Often, however, it is important to measure receptor phosphorylation, for which biochemical assays still trump the cell-based type. Minor said that bioluminescence resonance energy transfer has been used in cells to measure TK receptor phosphorylation, but only in low throughput. “We need a brighter fluorophore or better way to detect very low levels of fluorescence to measure receptor phosphorylation in intact cells,” she concluded.
Gareth Waldron, senior principal scientist in the ion channel pharmacology group at Pfizer, uses the ViPR voltage/ion probe reader from Aurora to conduct screening against ion channel targets in live cells. He said that the state of the cells and cell culture techniques are the keys to performing cell-based assays.
“You need proper confluence of the cells,” he said. “Also, dust control is a real problem. Dust fluoresces at a similar wavelength [to the fluorescent probes.]”
In a later discussion panel, John Dunlop, associate director of research at Wyeth stated that “it’s a must to use cell-based assays for ion channels. The platforms for screening ion channels are becoming much better validated, but it’s crucial to use an appropriate cell line.”
That sentiment was echoed by several scientists, who noted that cell-based assays clearly provide more relevant results, but if one isn’t using the right cell line, the results may not be much more relevant than those from biochemical assays. “It’s important to use human cells, because the receptors are often very different between species,” said David Monteith, research scientist at Lilly Research Laboratories.
For this reason, human primary cells are very desirable, but their use can’t always be justified. “Primary cells are very difficult and expensive,” Zaman said. “The goal often is to find a hit first, then use more complex primary assays later.”
One audience member asked a panel of pharmaceutical representatives what level of modification they could accept when using human cells, and how it affects the relevance of the assays.
“It’s acceptable to transform cells,” Dunlop said. “Everyone does it. We’ve spent a lot of time trying to use primary cell lines, but we’re not insisting on using them.”
Other problems commonly encountered in cell-based assays are associated with cell culture, including the natural variations that occur and bacterial contamination. “Maintaining cell culture puts a huge drain on resources,” said Peter Kotsonis, laboratory head of discovery technologies at Novartis.
For this reason, many pharmaceutical companies choose to leave cell culture to the experts. For instance, Peter Hodder, research fellow and head of HTS robotics at Merck, said that over 50 percent of high-throughput screening at his company is cell based; yet Merck contracts the cell culture out to a third party. “With every cell-based assay, something peculiar happens,” he said. “If you treat cells like a reagent, they’re one of the most unstable ones out there.”
Tools of the Trade
Instrument makers and vendors offered several solutions on the following day, such as technology designed to greatly reduce the number of cells and associated reagents necessary in an assay, or newer, more stable cell lines.
Seth Cohen, director of application sciences at Caliper, told an audience during a Caliper-sponsored luncheon that the newest version of the company’s LabChip series, the LabChip 3000, builds on its predecessor by incorporating two lasers, each of which is a better fit for commonly used fluorophores in cell-based assays. This, in turn, translates to a better fluorescent signal and fewer reagents.
He added that the system, which is not yet officially on the market, allows single-cell analysis, resulting in a 10- to 100-fold reduction in cell usage, and a 100- to 1,000-fold reduction in agonist usage. “It’s like flow cytometry, in that fluorescence from individual cells is recorded,” he said.
However the LabChip 3000 is not quite capable of the individual level of cellular analysis that flow cytometry can perform — Cohen said that typically the response of approximately 100 cells is averaged to yield a response for each cell line.
Other companies for whom reduction of primary cell usage and assay cost was a major marketing point included Cambrex and Vitra Biosciences. Dale Greenwalt, the director of assay development at Cambrex, discussed his company’s assays for human adipocytes, most commonly used in diabetes and obesity research.
Greenwalt said that although the cost of human primary cells is often cited as a problem, Cambrex’ AdipoRed assay reduces the cost to about 5 cents per well for a well plate-based assay. He also pointed out the silver lining in the variable nature of cell culture. “Variability of cells is not always a negative,” he said. “Lot-to-lot variability of cell culture is akin to patient-to-patient variability, [and is] something a lot of us want to look at.”
Simon Goldbard, the vice-president of product development in the cell biology section of Vitra, talked about the Vitra CellCard system, which consists of 500- by 300-micron chips on which primary cells can be grown to confluence for assay miniaturization. According to Goldbard, researchers can achieve confluence with 50 to 100 individual cells, resulting in up to a 100-fold decrease in cell usage without affecting data quality.
Also of note was a presentation by Andrew Parkinson, CEO of XenoTech. His company introduced a new immortalized human hepatocyte cell line — developed at MultiCell in Rhode Island — primarily for use in ADME/toxicity screening.
Hepatocytes are the gold standard for such assays, since most drug failures are due to liver toxicity. However, hepatocytes are often in short supply, and are some of the most variable cell lines in existence. (See related story in this issue: Geron, GXR, Roslin ...).