Automated Drug Absorption Assays

Drug absorption assays, making up the “A” in ADME/Tox testing, examine how a compound is able to cross various membranes in the body. One of the most important barriers is the intestinal membrane. In order for an orally administered drug to be able to affect its target area within the body, it must be able to pass from the intestine into the blood stream. Therefore, it is crucial to be able to assess compound movement in an accurate, easy to use in vitro format before testing in man during clinical trials.

Cell-based drug absorption assays, such as Caco-2 and MDCK are considered suitable surrogates for intestinal lining absorbtion of small molecule drugs. BioTek Instruments has partnered with Corning Life Sciences to develop automated drug absorption assays in a 96-well format. The assay uses HTS Transwell®-96 Permeable Supports from Corning, in conjunction with BioTek’s EL406 and Precision liquid handling instrumentation. All steps within the assay process were automated, including cell dispense, media exchange, plate washing, and compound dispense. The small footprint of the EL406 enabled the instrument to be easily placed within a laminar flow hood to ensure aseptic manipulations of the cells and media.

MDCK cells, expressing P-glycoprotein, were used as the model cell line for this application. Monolayer integrity was measured using Lucifer Yellow. Data from these tests show that little or no Lucifer Yellow was able to cross the cell monolayer from the apical chamber to the basolateral chamber, indicative of a consistently tight monolayer of cells being able to form within each well (Figure 1).

Drug efflux was also monitored using Rhodamine 123, which was placed into the basolateral chamber. This compound, being a substrate of P-glycoprotein, should be pumped through the cell layer into the apical chamber. Results from automated versus manual assay comparisons demonstrated that the automated assay showed higher efflux values due to lower variability across multiple replicates when compared to manual data (Figure 2).

Do you currently run your drug absorption assays in 96-well format? How do you feel automated high-throughput drug absorption assays can aid in your drug discovery process?

Cryopreservation of Tissue Culture Cell Lines

There are a number of reasons for the storage and preservation of tissue culture cell lines. Cell lines subject to continuous culture are prone to selective variation, senescence, as well as genetic and phenotypic drift. Despite the best practices, equipment failure and contamination can ruin culture stocks. Additionally the storage rather than the maintenance of unneeded cell lines can be savings of time and materials. For these reasons and more, cultured cells are typically stored “frozen” at very low temperatures for extended periods of time.

Optimal freezing of cells for maximal viable recovery upon thawing minimizes intracellular ice crystal formation. This is accomplished through the use of cryoprotectants such as DMSO or glycerol. Freezing cells at a high cell-concentration allows for sufficient dilution of the cryoprotectant upon thawing and reseeding, making centrifugation to remove the cryoprotectant unnecessary. It has been reported that optimal cell survival occurs if cells are cooled at 1°C/min [1]. This cooling rate is a compromise between fast freezing, which minimizes ice crystal formation and slow cooling, which encourages the extra-cellular migration of water.

Low temperature cell storage has a number of different options, each with advantages and disadvantages. The least expensive would simply be storage in a conventional -80°C electric freezer. Almost every cell biology laboratory has this type of freezer, making their use very appealing. Unfortunately the relatively high temperature of these devices, along with the continual opening of the access door to retrieve materials not slated for long-term storage makes these freezers less than ideal for long term viability. Loss of viability at this temperature has been estimated to be 5-10% per annum [2]. Dedicated ultra-low -135°C electric freezers provide a better option in terms of viability. These dedicated freezers do not “suffer” from being opened continuously. However they are mechanically complex, expensive to purchase and require some sort of liquid nitrogen back-up. As with any electric device, interruptions in power can be catastrophic without some form of generator or battery backup.

For most users, long term cryopreservation of cell stocks employs the use of liquid nitrogen storage. In regards to liquid nitrogen storage one can use a liquid phase or a vapor stage storage device. With vapor stage nitrogen storage vials are positioned above a shallow reservoir of liquid nitrogen, the depth of which needs to be carefully maintained. The advent of improved insulation and reduced evaporation, along with automated monitoring of nitrogen levels, has made this the preferred method of liquid nitrogen storage. In this type of storage a vertical temperature gradient exists in the vapor from -190°C upward. Depending on the containment vessel and the frequency at which it is opened the temperature gradient will vary. Because of the small amount of nitrogen used in this system close monitoring of the liquid level is essential. Liquid phase storage submerges the vials directly in the liquid nitrogen. This method, while requiring more nitrogen, provides a constant temperature of -190°C. The larger capacity of fluid in the system means nitrogen will last significantly longer than a vapor system. Because the cells are immersed in liquid, the potential for cross-contamination of samples, while remote does exist and has been reported. One significant danger comes from liquid nitrogen that has leaked into cryovials. It has the potential to become explosive when it is thawed.

With either nitrogen storage system there are several different freezer designs to choose from based on their opening or neck size and the storage system employed. The most popular are the narrow neck designs, which reduce the rate of evaporative loss of nitrogen, but make access more difficult. These types typically store vials in either a cane that is placed in round canisters or in a series of trays that hold storage boxes. In either case the canisters or trays are attached to a long handle that hangs from the top lip of the storage vessel. Wide mouth designs have a similar arrangement, but allow easier access at the expense of higher evaporation rates. Automated systems are also available where nitrogen liquid level is monitored and added from an external reservoir automatically.

On the whole, long term storage of cell lines is an expensive yet an absolutely necessary endeavor for those investigators running cell based assays. Liquid nitrogen is non-flammable, provides an ideal storage temperature, readily available from a number of sources, and relatively inexpensive. Care should be taken when using liquid nitrogen. Asphyxiation is a real hazard if Liquid N2 is being replenished in a closed area. Additionally liquid N2 is extremely cold, care should be taken to avoid frostbite.

What type of long term cell storage is used in your lab?

References

1. Leibo, S.P. and P. Mazur, (1971) The role of cooling rates in low-temperature preservation. Cryobiology, 8:447-452.
2. Green, A.E., B. Athreya, H.B. Lehr, and L.L. Coriell (1967) Viability of Cell Cultures Following Extended Preservation in Liquid Nitrogen. Proc. Soc. Exp. Biol. Med. 124:1302-1307.

Take3 Tips and Tricks for Micro-volume Quantification

The Take3 Multi-Volume plate has proven to be a versatile, well adopted product since its launch last year. Applications include native 260/280 nm absorbance micro-volume quantification of DNA and proteins as well as more sensitive quantification with fluorogenic reagents such as PicoGreen for dsDNA quantification.

Here are a few of the important considerations to keep in mind when working with the Take3 plate.

1. Clean slides ensure great data! Here’s a quick way to check the cleanliness of the slides: Put 2-3 µL of deionized, distilled H2O (ddH2O) on each spot and perform the blanking measurement with the Take3 interface in Gen5.
Use the “Use blank average” option in Take3 PreferencesCalculation and set the Validation Limit (%CV) to between 3 and 5 (whatever you are more comfortable with, the default is 10):




Read the plate. A clean plate should easily meet the limit. In the Applications Lab at BioTek Instruments, we routinely achieve CVs below 3% after thorough cleaning.

2. When working with extremely high concentrations of protein it may be necessary to clean the slide with a wetted wipe to remove any residual, precipitated protein. (This is most noticeable as streaking on the slide when wiping with a dry wipe). DNA does not appear to present this type of behavior even at very high concentrations.

3. Use a high quality multi-channel pipettor when possible to transfer samples, standards or reagents. This will help to eliminate some of the errors associated with multiple pipetting steps. Additionally, when working with any type of reaction chemistry on the Take3 Plate, the reagent addition should be done last and as quickly as possible to ensure equivalent kinetics across samples.

4. Gen5’s Take3 interface provides quick and easy nucleic acid and protein quant protocols, but the standard Gen5 protocol can also be used to define any number of micro-volume assays that would typically be performed in a microplate. The Take3 plate type is available in the Gen5 Plate database and can be selected to define a micro-volume protocol. Pathlength data can be easily entered into the data reduction steps using the values provided with each serialized plate.

5. Use replicate samples when possible. Replicate sample measurements can be used to identify flyers and ensure accurate results.

6. Change tips between transfers. The most accurate measurements will require consistent transfer volumes of the sample, standard or reagent. This is particularly important for assays that rely on reaction based chemistries. We have found that changing the tips often will help minimize pipetting errors.



It is best to treat the Take3 as you would any low volume transfer. Use the best pipettor for the task, preferably a recently calibrated 2 uL or 0.5-10 uL pipettor when possible. The use of low-adhesion tips can also improve the accuracy of transfer.

If you have further comments or questions or have developed your own assay for use on the Take3 plate we would like to hear them.

Automated Kd, Ki determinations for CXCR4-SDF1

The chemokine CXCR4 resides on some cell membranes and selectively binds the SDF1 ligand signaling a pathway that has multiple critical functions in normal physiologies including embryonic development of the cardiovascular, hemopoietic and central nervous systems. CXCR4 also plays a key role in breast cancer metastasis, and has been implicated as a co receptor in HIV-1 cell penetration, and a primary receptor for HIV-2 cell penetration. Its function in these and other disease pathologies are fueling the search for small molecule CXCR4 antagonists as a means for intervention (1).

The Product Specialist for the Cisbio Tag-lite assay line visited BioTek last week. He was here to collaborate with us on validating the automation of the ‘Chemokine CXCR4 receptor ligand binding assay’ using BioTek liquid handling and detection instrumentation. Using HTRF technology based on the competition between fluorescently labeled acceptor ligand and non-labeled compounds to bind with a CXCR4 donor labeled receptor site on a cell membrane, this robust assay offers a simple and confident way of determining CXCR4 antagonist properties of possible drug candidate compounds via Kd, Ki/IC50 and Z’ values. These values are commonly used to validate assay performance and determine binding affinity of known and unknown agonists and antagonists in protein-ligand reactions.

The assay is very user friendly and proved to be automation friendly as well. There are only a few distribution steps to the assay well: cells, compounds, and tracer. The cells and tracer are added to the wells in a single step using the same volume for all wells on the plate. The BioTek MicroFlo Select equipped with a 1 µl cassette was used for cell and tracer dispensing, and in the Z’ experiment also performed a simultaneous 4-compound dispense in addition to the cell and tracer (the Z’ test was fully automated using MicroFlo Select). Although the serial dilutions for the Kd determination and Ki/IC50 determination have different start volumes and dilution ratios, their end volumes are the same and they are both done as 11-point serial dilutions with a 12th point assay control and depending on the test were dispensed in either duplicate or quadruplicate to a 384-well low volume Greiner BioOne white plate. Automation of the serial dilution steps and loading the serial dilutions to the assay plate was done using BioTek's Precision XS. After all dispenses to the assay plate are complete, there is a 1-2 hour room temperature incubation step, followed by detection of the fluorescence intensity. An HTRF ratio of the fluorescence intensities was used to calculate Kd, Z’, and IC50/Ki values. Data generated by automating the assay to that generated by performing the assay manually was used to judge viability of the instrumentation in performing the assay. The results of this successful collaboration will be highlighted by both an upcoming Application Note and a poster presentation at the 2010 MipTec conference in the early fall.

[1] H. Tamamura et al. (2008). “A future perspective on the development of chemokine receptor CXCR4 antagonists”, Expert Opinion on Drug Discovery, 3(10), pp. 1155-1166.

Biofuels – The Importance of Algae

Diminishing stocks of fossil fuels along with the increasing emission of greenhouse gases such as carbon dioxide as a result of fossil fuel combustion has driven the research on alternative and renewable fuel sources such as biomass-derived fuels. Microalgae represent a potential source of biomass fuel production. Microalgae is a generic term describing small prokaryotic (cyanobacteria) and eukaryotic organisms found in most freshwater and marine systems that use sunlight to fix carbon by making sugars from CO2 and water.

Unlike grain based ethanol production algal fuel production does not compete directly with food stocks, making their use more palatable. Many microalgae have the ability to produce large amounts of triacylglycerols (TAG) as a storage lipid under certain stress conditions. Unlike terrestrial plants, microalgae do not require fertile land or irrigation. Because algae consume carbon dioxide, large scale cultivation can be used to remediate the combustion exhaust of power plants. Unfortunately most of the cultured strains of photo-synthetic micro-organisms used in the laboratory were selected for their ease of cultivation or as genetic modeling systems rather than their ability to produce biofuel compounds [1].

Recent work has focused on improving lipid content of algae by identifying improved growth media, isolation of specific over-producing strains or through the use of genetic modification of microalgal metabolic enzymes [2]. Because of the large number of growth variables including light intensity, ionic strength, dark cycle length, pH, temperature, culture dish geometry, CO2 concentration, mixing, nitrogen and phosphate concentrations, water purity, media feedstock, etc., fractional factorial experimental designs [3] are often used to measure many factors in a manageable number of experimental runs. Microplates (96‐ and 384-well) make perfect culturing vehicles for assessing growth and/or TAG production from algal cultures under these numerous experimental conditions. The multimode readers from BioTek, such as the Synergy H4, Synergy 2, and Synergy Mx are ideal reader platforms for quantitation of these metrics and the many others associated with biofuel production.

References

1. Hu, Q., M. Sommerfield, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins (2008) Microaggal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The Plant Journal 54:621-639.

2. Rosenberg, J.N., G.A. Oyler, L. Wilkinson, and M.J. Betenbaugh (2008) A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr. Opin. In Biotechnology. 19:430-436.

3. Montgomery, D.C. (2001) Design and Analysis of Experiments, 5th ed. Wiley

Drug-Drug Interactions 2010 Conference Summary

The 13th International Conference on Drug-Drug Interactions (DDI) was held in Seattle, WA this past week. The conference was a focused symposium examining the current scientific concepts and practices in DDI evaluation. Topics covered included inhibition and induction of drug metabolizing enzymes, especially Cytochrome P450s, and drug efflux and uptake transporter enzymes. The conference had approximately 120 attendees spanning academia, government regulatory agencies (FDA), the pharmaceutical industry, and tool providers.


A pre-conference workshop was held, which included a session concentrating on “Higher-Throughput Evaluation of DDI”. During this session a talk was given by BioTek accentuating the project work that has been done to demonstrate the utility of our instruments to be used for automated multiplexed cell-based Cytochrome P450 assays. While not all of the industry may be ready to move into a higher throughput format, many attendees seemed interested in the type of instrumentation that BioTek had to offer for these types of applications.


The remainder of the conference focused on why DDI studies are performed, and the enzymes that play a role in drug absorption, distribution, metabolism, and excretion (ADME), that when inhibited or induced can lead to potential adverse drug-drug interactions. The goal was to move towards determining the best techniques for examining the ADME of new chemical entities (NCE), as well as how these potential new drugs may affect the efficacy of other co-administered drugs. It was clear, by the end of the conference, that even though the number of adverse drug-drug interactions being reported in the literature is decreasing, there is still much work that needs to be done to increase the confidence in the results that are being published, as well as being placed on the labels of prescriptions in your medicine cabinet.

ROS Revisited

Reactive oxygen species have become an important topic in regards to normal cellular function and their potential for carcinogenesis. Originally ROS were only thought to be released by phagocytic cells as a part of the host defense response, now it is clear that they have a role in a number of normal cellular processes. Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades [1]. It should be noted that ROS can serve as both intra- and inter-cellular messengers. The short half life of most reactive oxygen species makes them an idea intracellular messenger.

As cells proliferate, they move through a coordinated process of cell growth, DNA duplication and mitosis referred to as the cell cycle. The cell cycle is a tightly regulated process with several checkpoints. Each one of these checkpoints is regulated by proteins and protein complexes that are influenced by the oxidative state of the cell. The relationship between the Redox state and cell cycle control is described in great detail in a review by Burhans and Heintz [2].

High levels of ROS, which can lead to cellular damage, oxidative stress and DNA damage, can elicit either cell survival or apoptosis mechanisms depending on severity and duration of exposure. Intra-cellularly, ROS species, in conjunction with antioxidant enzymes, are believed to play a role in turning enzymes on and off by redox signaling in a manner akin to that of the cAMP second messenger system [3]. Stimulated cells exhibit pronounced increases in ROS activity.

Figure 1. DCF Fluorescence in Stimulated Cells Measured over Time.

The integral role that ROS compounds have been shown to play in cellular growth and multiplication has makes them and/or the enzymes that produce and regulate their production potential chemotherapeutic drug targets. In addition the general redox status of the cell has been suggested to act as a cellular growth control mechanism. As cancer cells seem more susceptible to perturbations in the cellular redox-state, therapeutic agents that alter it have the potential to be effective antitumor agents.

Several different fluorescent and luminescent markers have been developed to assess ROS, particularly H2O2 in cells and tissues. The multimode readers from BioTek, such as the Synergy H4, Synergy 2, and Synergy Mx are ideal reader platforms for quantitation of these compounds and many others associated reactive oxygen species.

References

1.Hancock, J.T., R. Desikan, S.J. Neill, (2001) Role of Reactive Oxygen Species in Cell Signaling Pathways. Biochemical and Biomedical Aspects of Oxidative Modification, 29(2):345-350.

2.Burhans, W. and N. Heintz (2009) The Cell Cycle is a Redox Cycle: Linking phase-specific targets to cell fate. Free Radical Biology and Medicine. 47:1282-1294.\

3.Hou Y.C., Janczuk A. and Wang P.G. (1999) Current trends in the development of nitric oxide donors. Curr. Pharm. Des. June, 5 (6): 417–471.