Generation of primary hepatocyte microarrays by piezoelectric printing

We demonstrate a single-step method for the generation of collagen and poly-l-Lysine (PLL) micropatterns on a poly(ethylene glycol) (PEG) functionalized glass surface for cell based assays. The method involves establishing a reliable silanization method to create an effective non-adhesive PEG layer on glass that inhibits cell attachment, followed by the spotting of collagen or PLL solutions using non-contact piezoelectric printing. We show for the first time that the spotted protein micropatterns remain stable on the PEG surface even after extensive washing, thus significantly simplifying protein pattern formation. We found that adherence and spreading of NIH-3T3 fibroblasts was confined to PLL and collagen areas of the micropatterns. In contrast, primary rat hepatocytes adhered and spread only on collagen micropatterns, where they formed uniform, well defined functionally active cell arrays. The differing affinity of hepatocytes and NIH-3T3 fibroblasts for collagen and PLL patterns was used to develop a simple technique for creating a co-culture of the two cell types. This has the potential to form structured arrays that mimic the in vivo hepatic environment and is easily integrated within a miniaturized analytical platform for developing high throughput toxicity analysis in vitro.

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6 mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r ≥ 0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 μg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.