Cellular World Poised for Change

Cells offer the first level of structural and functional organization in any biological system. Although they exhibit the complexity of interacting networks, cells are relatively simple to use and hence it’s no surprise that they are used so extensively all through the pharmaceutical drug pipeline. Cell-based assays are used in early discovery for screening drug candidates and later in development for toxicity testing. Cells are further used in manufacturing antibodies and other biologically derived drugs, and also used as therapeutics in regenerative medicine.


Informative Screening

Up until the mid-’90s, 80-90% of assays used for screening drug candidates were in vitro biochemical assays. “It was all biochemical, very simplistic and uni-dimensional,” says Lisa Minor, PhD, principal scientist in the secondary pharmacology group at Johnson & Johnson Pharmaceutical R&D in the US. “It was very straight-forward because you would be interacting with a target with a known binding site and crystal structure.” Cell-based assays were used but mostly as a follow-up in secondary screening if no in vitro assays were available.

The use of cell-based assays rapidly increased as imaging tools to probe their biology became available. The first breakthrough in cellular imaging was the introduction of the Fluorometric Imaging Plate Reader (FLIPR) by Molecular Devices Inc. for monitoring the activity of G- protein coupled receptors (GPCRs) and ion channels. FLIPR measured changes in intracellular calcium in real-time and in a high-throughput screening (HTS) environment, thereby directly linking the activity of the drug to the biochemical changes taking place in the cell.

“The important thing that it did was get the pharma industry thinking about the use of cells,” says Lansing Taylor, PhD, CEO of Cellumen Inc., a company based in Pittsburgh, USA. Taylor was formerly affiliated with Cellomics Inc., a company that pioneered the use of high-content screening. Soon pharmas began to appreciate that although complicated and low-throughput, compared to biochemical assays, cell-based assays provided more biologically relevant information. “Cell-based assays are now the largest part of discovery, averaging about 60-70 % and its use is increasing in development,” says Taylor.

Physiological Relevance

Minor’s responsibilities in the secondary pharmacology group at J&J are to put into practice the “fail early, fail cheap” mantra that is touted by the pharmaceutical industry. She is working to develop a panel of tests to be able to predict if a compound will succeed later in development. For instance, she is hoping to develop a cell-based assay using a panel of receptors to screen a series of lead compounds. “What the test will tell you is whether or not your compound inhibits or activates any of the receptors that are being screened,” according to Minor.

“Then you have to ask the question as to what is the liability of hitting that receptor and what is your safety margin?” Currently such assays would be done using cell lines. “What would be better is if we could do the same thing in a primary cell or in a cell with more primary characteristics,” she says.

Cellumen Inc. has put together such a panel in its CellCiphr system in order to offer more physiologically relevant data and better predicitivity. The system brings together a combination of human cell types - such as hepatocytes, renal cells, neuronal cells, cardiomyocytes and immune cells - coupled with the right decision-making software, to create an effective predictive tool.

“The key is the selection of the right cell type, the right panel of functional biomarker read-outs and the construction of the classifier based on compound libraries with known safety data,” says Taylor. The panel is currently designed in a 384 well format and measures 11 different cellular parameters at 3 time points with 10 point dose response curves, compatible with any high content imaging platform. Although the first generation panels use cell lines, Taylor is hoping to use more primary cells and tissue-engineered arrays in the creation of the next generation panels.

However, one of the problems with using primary cells is that they cannot be expanded to get enough cells for HTS. There is also a lot of donor variability, in terms of how they respond functionally. Some of that is genetic while some of it depends on the state of the cells when they are isolated. Stem cells are now generating a lot of interest as a renewable, scalable source of primary cells.

“With stem cells you can achieve genetic homogeneity, if you want it, but because you can isolate them from a number of different donors, you can also cover the spectrum of genetic variability in a controlled way,” says John E. Hambor, PhD, CEO of Cognate Bioservices Inc., a company based in Connecticut, USA, that works with pharmaceutical clients to design and develop stem cell-based screens. The company obtains tissues such as liver, brain, cord blood, umbilical cord, and bone marrow from various Organ Procurement Organizations (OPOs). The stem cells derived from these sources are then used to generate various types of cells such as adipocytes, dendritic cells, myocytes and others.

Hambor started looking into stem cell-derived cells for screening as an alternative to transformed cell lines while he was still a scientist at Pfizer. “We were beginning to appreciate that the number of compounds that were being discovered in these artificial cell systems were irrelevant and that led to a lot of work in sorting to find the really promising compounds versus those that were artifacts from the screening system,” says Hambor. Stem cells offered a more physiologically relevant in vitro model predictive of organ physiology and while the screens resulted in fewer hits they proved to be more suitable as drug candidates.

Another promising source of cells is the embryonic stem cell. Cellartis AB is a biotechnology company based in Sweden and the UK that is focused exclusively on the use of human embryonic stem cells (hES). The company has developed nearly 30 different hES cell lines and is using them to generate two specific cell types - hepatocytes and cardiomyocytes.

“The reason for choosing these two cell types is that there is a huge need for functional hepatocytes and cardiomyocytes in the pharmaceutical industry for drug testing,” says Peter Sartipy, PhD, senior scientist and project manager at Cellartis. “So far there is no real evidence that adult stem cells can spontaneously differentiate into hepatocytes and cardiomyocytes that the embryonic stem cells do. Most populations of adult stem cells also have a limited capacity for self-renewal.” Cellartis is also exploring the use of undifferentiated stem cells for drug testing.

“The limitation right now is that most pharmaceutical companies do not have individuals that have the skill set to propagate stem cells,” says Hambor. “It’s a little bit more complicated than doing normal cell culture. These cells are very sensitive to culture conditions and tend to differentiate.” Hence, many pharmaceutical companies are seeking external collaborations with companies that provide such expertise rather than having to develop the skills themselves.

Assays On Demand

Irrespective of their type and source, culturing cells tends to be a tedious activity that requires a lot of time, effort and attention to detail. According to Priya Kunapuli, PhD, senior research fellow in the Automated Biotechnology group at Merck & Co. in the US, the amount of time and effort that goes into the day-to-day care and maintenance of a live cell line is huge. “If you can buy the cells as a reagent and use it in the assay, you have cut out at least 30% of your work right there,” she says.

Kunapuli started evaluating the use of frozen cells as a biochemical reagent to be used on an as-needed basis, back in 2003. “We wanted to uncouple the production of the reagent, in this case the live cell, from the robotic screening so that we could have a just-in-time scaled up reagent, with the flexibility of scheduling when you want to do it and how much you want to do.” It also eliminated problems associated with human variability, scalability and homogeneity of cells. She started testing various technologies and methodologies to produce functionally stable and consistently performing frozen cells. Today about 25% of the company’s cell-based assays are done using frozen cells. “We have been continuing to use frozen cells on a case by case basis, depending on the biology,” she says.

Cellular Innovations

However, cells do have limitations in terms of use. They have limited use in screening certain types of drug targets, like ion channels. They cannot be used for bioavailability testing, and cellular results do not always correlate with what happens in vivo. However, there are also a lot of improvisations taking place to overcome these limitations. There is innovation in cell culture growth media and matrices to recapitulate a more physiologically relevant environment.

The creation of 3D cell cultures using complex media and structures like beads rather than flat plastic surfaces to grow cells is an attempt to get closer to replicating the complex organization and function of interacting cells. Label-free imaging technologies are also gaining hold as they offer a way to study the biology by minimally perturbing the system.

Automation is also enabling the growth of many different cell lines simultaneously and in a miniaturized format to minimize variability. Sophisticated informatics tools are now available to readily turn data into knowledge and help decision-making. While most drug companies are still in a wait-and-see mode keeping a close eye on these new technologies as they mature, there is no doubt that there will be a number of changes in the next couple of years on how things are done in the cellular world.