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Feb 1, 2012 (Vol. 32, No. 3)

Discovery Aided by Complex Assays

  • The Power of Co-Cultures

    Click Image To Enlarge +
    Tumor cell fibroblast co-culture showing tumor cell induction of a-SMA. Tumor cells are stained blue. The activated fibroblasts are stained green. Fibronectin is stained red. Lung tumor cells are shown co-cultured with normal human dermal fibroblasts. [AstraZeneca]

    According to AstraZeneca’s Dr. Barry, there can be a high stromal fibroblast content in colorectal and lung tumors, which can play a significant role in drug resistance.

    “If we model our tumor cells with fibroblasts, this will allow cross-talk and help to develop more realistic platforms to assay compounds,” said Dr. Barry, whose group showed that PK1D tumor cells grown on fibroblasts in co-culture formed a ball and the proliferation pattern of the cells changed.

    Using a PLA, he also showed the MAPK/ERK kinase (MEK) inhibitors and drug sensitivity profiles changed.

    “In this co-culture environment my dose curves alter by around five- to tenfold and this is perhaps more accurately modeling what is happening in a real tumor,” summarized Dr. Barry. “In fact, for tumors, I believe we should be looking at utilizing these co-culture systems before we take compounds into animal studies.”

  • Ultimate Challenge

    Click Image To Enlarge +
    Histological image of the HaCat human keratinocyte cell line growing in 3-D throughout the alvetex scaffold. The culture was fixed, wax embedded, and sectioned in the transverse plane and subsequently counter-stained (H&E). [Reinnervate]

    One reason 3-D cell culture is not yet widely adopted is that there are very few 3-D scaffold options that have been successfully commercialized, noted Dr. Przyborski from Reinnervate. He discussed the use of alvetex®, a polystyrene scaffold of interconnected voids in which cells can enter and grow to form complex 3-D structures.

    Dr. Przyborski presented data on hepatocyte cells grown in alvetex that showed that the cells were forming bile canaliculi, producing albumin, and also had elevated levels of a cytochrome p450—Cyp3A4—demonstrating that in the 3-D culture the cells had functionality that more closely mimicked real liver tissue.

    After discussing data that showed that skin cultured in alvetex formed keratinocytes and cornified envelopes indicating the formation of a stratum corneum, he also pointed out that human pluripotent stem cells cultured in alvetex formed neuronal rosettes and 3-D neural networks.

    “Alvetex is available as well inserts for 12- and 6-well plates, as well as deep-well plates,” said Dr. Przyborski. “Petri dish formats and a 96-well inset will be available in 2012. Scientists can easily reconstruct the 3-D environment for cells with alvetex and it will make 3-D culture as easy and routine to perform as traditional cell culture.”

  • Technology Gap

    The speakers all cited either throughput or cost as barriers to the use of complex cell assays for routine screening. As Dr. Ludbrook noted, “At GSK we like cost-effective, fast assays. Some of the more complex cell-based assays using techniques such as flow cytometry are just too low throughput.

    “We use flow cytometry in a kinase program to support lead optimization but can only characterize a fraction of the number of compounds we need to. This is why we’re investigating using the HyperCyt® technology for flow cytometry, as this will take us into real-world screening throughput because it reduces plate read time from 120 minutes to 14 minutes for a typical assay, and therefore gives us the potential to screen thousands of compounds in a disease relevant assay."

    “Primary cell assays are expensive,” added Dr. Bays. “With whole blood, you can only get so much blood to work with from one draw and it requires special handling conditions. We use instruments like the Labcyte ECHO to reduce volumes as much as possible, but it still currently costs us about $900 per run for antibodies alone.

    “We have not yet reached our goal of spending less than $1 per well. However, we have reduced costs significantly and are not spending anywhere near the $23 per well cost we incurred when we were first developing these assays and had to rely on standard reagent sizes.”

    According to Dr. Barry, qPCR is too expensive at the moment for the PLA “but as qPCR and microfluidics are on the point of explosion, it may get to the cost point where we may in the future be able to use this technology for transcriptional screening.”

    Dr. Ludbrook identified a wider scientific gap in the understanding of toxicity readouts compared to efficacy readouts.

    “The battery of assays used in understanding toxic characteristics of compounds should lead to a reduction in attrition at this stage of drug discovery,” he said.

    “However, there is less understanding of translational complex cellular assays that are truly representative of disease. The risk is we could be taking a larger number of safer candidates into expensive and time-consuming Phase II/III trials.

    “But if we fail to improve our predictions of efficacy, then we will be failing later and more expensively. We need to focus on this to enable us to bring more medicines to patients at a lower cost of development.”

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