Maximising the use of multiwell assays

Sara Verna discusses trends and challenges in microplate development, why cell based assays are more biologically relevant than other assays and common applications with cell based assays.

According to the latest study of R&D productivity by Deloitte and Thomson Reuters, from 2011 to 2012, the cost of bringing a new drug to market increased by approximately 30%.The authors estimated that the cost to launch a new drug is approximately $1.1 billion (€800 million) and that greater than 50% of the cost of drug development is attributable to the high expense of clinical trials. 

Failure at the clinical trial stage is extremely expensive for a company, and collecting information on how drug candidates interact with cells early in the discovery process will help identify prime candidates to take to clinical trials. 

Cell-based assay use is rapidly increasing in pharma and academic research. The cell based assay global market is expected to grow US $1.5 billion (€1.1 million) by 2017. In the first stage of drug discovery, basic research involves identifying cellular and genetic targets. 

Cell-based assays are more biologically relevant than biochemical assays because they provide an indication of in vivo drug activity in an in vitro setting.

In addition, collecting information on how drug candidates interact with cells early in the discovery phase will help avoid the high cost of late stage failures. Cell-based assays provide high-throughput screening of targets and functional experiments that target binding and specificity. It is estimated that microplate cell-based assays account for half of all assays used in drug development for primary and secondary drug screening and toxicity testing.

Due to increased utilisation of cell-based assays in pharma and academic research, demand for microplate readers with superior imaging and analysis for fluorescence signals is growing.

Microplate reader manufacturers have advanced technology to detect signals originating from a living cell through design of features for optimal temperature, gas and pH environments combined with enhanced direct optical approaches in bottom reading. Researchers should be aware of new equipment with two important new technologies that can help prevent background issues seen in some of the most precise readers.

First, interference from phenol red in cell media requires an optical feature that minimises auto-fluorescence from cell media. Second, since adherent cells do not attach homogeneously at the bottom of each well, it is important to purchase a microplate reader model that has optical features that detect signals of each well and the entire plate.

The issue of even distribution of cells at the bottom of the wells is a common problem in microplate-formatted cell assays. The term ‘edge effect’ refers to the observation that results measured from the wells on the edge of the plate may often be statistically different those collected from wells towards the centre of the plate.

Values can vary, and sample results obtained from the edge wells may be higher or lower that those towards the centre.

There are methods to prevent the edge effect. These include: covering plates during incubation to prevent evaporation, evaluating the assay workflow environment and limiting plate movement.

Another common practice researchers perform to maximise even distribution of cells is to allow the plate to sit for one hour at ambient temperature prior to placing it in 37°C incubator. This will allow the cells to distribute more evenly at the bottom of the wells.

Accurately controlling temperature is crucial to maintaining cell viability. Throughout the assay it is important to avoid moving the plate in and out of incubator since this can cause the edges of the plate to heat or cool at different rates from centre. It is also important to assess the lab space and general area to minimise temperature gradients or other environmental factors.

Also, laboratory personnel may be a source of contamination and human error is a factor known to enable contaminants to enter a cell culture.

Preventing evaporation with plastic lids that have condensation rings or breathable seals that allow gas exchange is an important consideration.

The Thermo Scientific Nunc Edge Plate with evaporation barriers allows researchers to use all 96 wells without exclusion of outer rows due to concern about edge effect. This product has superior optical properties for fluorescence signalling in microplate readers.

Demand is increasing for features that will maintain cell viability, keep samples free from contaminants and that are easy to use. Manufacturers have addressed usability by designing microplate readers with features like a user interface with multiple language capabilities.

Operator log files in the software and acoustic alert features help managers monitor personnel usage and sample safety. High sensitivity, a wide dynamic range and low cross talk ensure accurate and precise results for 96- to 384-well plates.

To generate the ideal physiological environment for live-cell assays, high performance microplate readers allow control and monitoring of CO2 and O2 levels to provide the flexibility needed for a busy lab. These sophisticated instruments have dual sensor modes for CO2 and O2 concentration as well as manual modes to monitor levels of premixed gases.

Superior cell growth is only possible in a sterile environment with constant atmospheric conditions. The advantage of incubating and measuring in one combination device is the driving force behind new techniques in multiwell assays.

There are many contaminants in labs, and having samples incubating and measured in the same microplate reader minimises the exposure to atmospheric impurities. This feature can also help avoid excessive movement of plates and accidental errors. In addition, throughout the cell assay process, exact monitoring of growth curves with no gaps in data can be optimised with a combination system.

Once the growth process is optimised, detecting metabolic changes with multimode and absorbance readers in the same instrument helps streamline the research process.

There are many cell-based applications that utilise microplate readers. This widely used instrument enables multiple samples to be measured in long-term proliferation and induction studies, growth monitoring of multiple different microorganisms and high throughput screening of proteins in cells. For example, when using a Nunc plate that minimises evaporation, data can be collected in the same instrument, and cells can incubate for long term studies. Another important application used in microplate readers is measuring proteins in carcinoma cells after hypoxia induction.

An application for growth monitoring of bacteria, such as the study where Helicobacter pylori O2/C02 regulation is measured for 28 hours is now possible with new technology.

Multimode microplate readers are rich with innovative features designed for the most widely used applications for 96 and 384-well plates. Whether detecting reporter genes, proteins in cell signalling, or metabolic processes, these instruments now have user-friendly platforms and optical features with excellent levels of sensitivity for fluorescence or luminescence-based assays to support research centres.  

A clear understanding of the challenges in cell-based assays and instrumentation features designed to control common problems will help researchers identify prime target candidates.

Sarah Verna is Product Manager – Cell Culture, Thermo Fisher Scientific.

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