New, innovative solutions in cell biology are constantly being developed to support biological research, and can be applied to the drug discovery process. However, in order to convince the scientific community of the promises these applications offer, they need to be validated under real world conditions. This can be done through independent and unbiased life science organisations with expertise in biology. Read our blog below to learn about one such innovative technology that was validated in our hands.
Cryopreservation is the most common way to preserve cells for shipping. However the process requires either storage in specialist and expensive media or more commonly in a mix of foetal bovine serum and DMSO and whilst the cells survive on dry ice or liquid nitrogen some cell types behave differently once retrieved. This impedes progress of research projects and also results in high shipping costs. A relatively new and innovative technology that can potentially overcome these limitations is a product called ‘WellReady™’ by Atelerix. When added to media the WellReady™ reagent polymerises to form a semi-solid gel on and above the cells and maintains the cells in a quiescent state when shipped between 10 and 20oC over a five-day shipping period or longer. This gel can be de-polymerised after shipping with the addition of other reagents and, following a media change and overnight incubation, the cells can be used as an adherent monolayer.
The advantages of this approach are:
- Cells do not need to be cryo-preserved before shipping, saving costs and time/difficulty to resurrect cryo-preserved cells
- Cells can be grown at source and shipped as adherent confluent monolayer in micro-well plates, then used at the destination with minimal manipulation
- Cells can be shipped optimally at temperatures of 10-20oC, therefore dry ice or wet ice are not required, reducing shipping costs and paperwork
- Cells maintain transient expression of proteins even after 5 days in gel, suggesting this approach could ‘pause’ a batch of cells in plates allowing the operator to transfect a batch of cells, plate out and assay on a day by day basis
In this case study, the quality of cells preserved and shipped in WellReady™ was evaluated using the following assay formats:
Calcium flux based FLIPR assay
FLIPR Calcium Assays are a popular technique used for the assessment of calcium flux in high and ultra-high throughput formats generally associated with G-Protein Coupled Receptors (GPCR’s). The assay uses a monolayer of cells expressing the target of interest, media is replaced with buffer containing a calcium sensitive dye that is absorbed into the cell’s cytoplasm during incubation. Ligand-receptor binding triggers the release of intracellular calcium, which in turn binds to the calcium-sensitive dye, thereby increasing fluorescence intensity. FLIPR uses an extracellular masking technique to block background fluorescence and increase the assay’s signal window.
NanoBRET™: A live cell kinase assay
Promega’s NanoBRET™ Target Engagement is a first of its kind assay that enables researchers to quantitatively measure test compound binding to full-length kinase targets expressed in live cells. This analysis is be performed in cultured cells using a technique known as Bio Resonance Energy Transfer (BRET) that occurs between a 19-kDa Nanoluc luciferase tagged target kinase and a cell-permeable fluorescent energy transfer tracer probe introduced into the assay that binds specifically with high affinity to the kinase. When the fluorescent molecule comes into close proximity with the Nanoluc enzyme in the presence of substrate the resultant photons activate the fluorophore to generate luminescence. Addition of competing compounds results in displacement of the tracer and subsequent dose-dependent decrease in NanoBRET™ signal, which allows quantification of the intracellular affinity of the test compound for the target kinase.
CyclicAMP-response element (CRE) based luciferase assay
CyclicAMP-response elements or CREs exist in the promoter region of many genes known to play important role in metabolism, neurotransmission, development, cell cycle, memory formation, etc. This Luciferase Reporter Assays detects changes in cyclic-AMP within the cell allow investigation of the promoter activity by measuring light output from luciferase enzyme that is expressed under the control of the promoter of interest. These assays are generally rapid, sensitive, easy to perform and cost-effective.
Results from these three quite different assays suggested that cells maintained in WellReady™ during shipping over a 5 days period responded in a similar manner to stimulation compared with cells that had not been shipped. This raises the possibility of shipping other cell types such as primary cells and differentiated stem cells in well plates in a manner such that, on arrival, cells could be cultured for 4 to 24hrs then used for bioassays in a quick and convenient manner.
Are you working on a novel approach that could be applied in drug discovery research? Aurelia Bioscience, a UK-based contract research organisation (CRO) uses decades of scientific expertise to validate new technologies and deliver benefits in biomedical research. Get in touch to find out more!