BioFiles Volume 5, Number 8 — Cell-Based Assays

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Introduction

Don Finley
Don Finley

The challenge of understanding the biology of living organisms, including disease processes, is the incredible complexity involved. Scientists early on realized the value in utilizing life's most fundamental unit, the cell. Many aspects of living organisms are analyzed based on studies of living cells from cell signaling, proliferation, apoptosis, cellular genetics/morphology, neurobiology, cancer and more.

In both the pharma industry and academic research, the term cell-based assay is commonly used to refer to any assay based on some measurement of a living cell.

The pharmaceutical industry's need to efficiently commercialize drugs is a driving force in cell-based assay innovation. The industry is hoping to utilize cell-based assays to help reverse the increasing trend of costly late-stage drug failures. Having an Investigational New Drug (IND) candidate fail in phase III clinical trials can result in a loss of several hundred million dollars for that company. It is reported that with the current level of late stage failures the return on investment for pharmaceutical companies could drop as low as 5%, which is an unacceptable level for most companies.

A lot of scientific resources are being poured into cell-based methodologies. The promise these technologies need to fulfill is daunting and the phrase “fail early” is an over-simplification. There is a need for rapid cell-based methodologies to predict with a high degree of accuracy which IND will be successful through to commercialization.

Drugs are failing for a number of reasons. While animal models are commonly used for toxicity and metabolism studies of INDs, they are both expensive and low-throughput which usually limits their use to late stages of preclinical testing. Additionally, animal models may not always replicate human metabolism which results in late-stage failures due to unforeseen toxicity issues. Adverse drug reactions (ADRs) due to idiosyncratic differences in humans are another cause of late-stage drug failures. For example, Troglitazone, a type II diabetes drug, was taken off the market 3 years after release due to a small number of cases of drug-induced hepatitis. ADRs due to idiosyncratic differences tend to be rare events and even large clinical phase III trials could fail to detect the low rate (about 1 in 10,000) of idiosyncratic liver failure typical of this type of safety issue.

Many cell-based methodologies are being considered but so far no single technology or group of technologies has been reported to improve the late-stage failure rate. One important consideration is the cell type used for the metabolic and toxicity testing. Primary cells are often preferred over longterm established cell lines because they are thought to more closely replicate “real cells”. However, primary cells are variable and often can only be cultured for a few passages before they reach senescence or undergo undesirable phenotypic changes. Established cells have the benefit of phenotypic stability and ease of culture and are used extensively for cell-based assays. For example, cell lines like HepG2 and NIH/3T3 are ideal for testing mitochondrial toxicity because of their anaerobic metabolism. However, in regards to liver toxicity studies, established hepatocytic lines are less than ideal because they only weakly express cytochrome P450 enzymes.

Current research is investigating the use of induced pluripotent stem cells (IPSCs) for creating better in vitro models for drug studies. For example, differentiated stem cells may be better models for studying drug-induced liver injury than primary hepatocytes as they maintain function longer than primary cells. ADRs due to idiosyncratic events are often due to patientspecific susceptibility factors such as genetic variantions. One proposal to address this issue is to create large libraries of IPSCs that can be differentiated to hepatocytes as the basis for an automated assay.

Another limitation to traditional cell-based assays is a geometric issue. Most cell culture assays are performed in two dimensions while the real world of an in vivo cell is three-dimensional. 3-D cell culture can better create or mimic the extracellular environment (geometric shapes, scaffolding, forces placed on cells) so that cells will react to stimuli, toxins etc. more like they would in vivo.

A broad offering of products from Sigma® Life Science are used in cell-based assays that are suitable for both pharma and academic applications. We have broadened our U.S. offering of established cell lines and hybridomas to include over 1500 cell lines from the European Collection of Cell Cultures (ECACC®). ECACC cell lines are guaranteed to be authenticated and mycoplasma free.

We have the best selection of reagents and biologically active compounds in the industry. Our LOPAC® library makes accessing these compounds far easier than ever with thousands of pre-dispensed compounds on 96-well plates. In addition, we offer an exhaustive collection of Prestige Antibodies® for detection of a wide variety of cellular organelles and gene products that serve the basis for a broad variety of cell-based assay applications.

Sigma has applied the revolutionary CompoZr® Zinc Finger Nuclease technology to create an unparalleled range of genetically modified mammalian cell lines for use in areas such as basic research, target validation, drug discovery and drug development. With targeted and heritable gene deletions, integrations, or modifications our isogenic cell lines give you the tools to take your research to new heights. In the coming months you can expect a number of cell lines that will enable cell-based assay innovation never before possible.

References

  1. Tsaioun, K., Bottlaender, M., Mabondzo, A. ADDME - Avoiding Drug Development Mistakes Early: central nervous system drug discovery perspective. BMC Neurol. 9, S1–11 (2009).
  2. Gilbert, J., Henske, P., Singh, A. Rebuilding Big Pharma's Business Model. The Business & Medicine Report. 21 (2003).
  3. Shaw, R. Industrializing Stem Cell Production. BioProcess Int. 8, 10–15 (2010).

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