Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip

Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid–hydrogel model that better mimics the cell–cell and cell–extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.

High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells

To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100μm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.

Analysis of 8000 proteins and reduced carry over significantly increase the throughput of single-shot proteomics

In the field of LC-MS based proteomics, increases in sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of data that can be generated do however also depend on the performance provided by nano liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be of paramount importance to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. As an alternative to traditional packed bed LC column technology that uses beads packed into capillary tubing, we present a novel LC column format based on photolithographic definition and Deep Reactive Ion Etching (DRIE) into silicon wafers. With a next generation pillar array column designed for universal use in bottom-up proteomics, the critical dimensions of the stationary phase support structures have been reduced by a factor of 2 to provide further increases in separation power. To demonstrate the potential for single-shot proteomics workflows, we report on a series of optimization and benchmarking experiments where we combine LC separation on a new generation of pillar array columns using Vanquish Neo UHPLC with fast Orbitrap Tribrid MS data-dependent acquisition (DDA) and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). In addition to providing superior proteome coverage, robust operation over more than 1 month with a single nanoESI emitter and reduction of the column related sample carry over are additional figures of merit that can help improve proteome research sensitivity, productivity and standardization.