Microfabricated Arrays for Splitting and Assay of Clonal Colonies

A microfabricated platform was developed for highly parallel and efficient colony picking, splitting and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 μm diameter and 100 μm length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78–92%. To demonstrate the technique’s utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only 5-man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34-man hours expended over 47 days. These data support the considerable reduction in time, manpower and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches

Transparent magnetic photoresists for bioanalytical applications

Microfabricated devices possessing magnetic properties are of great utility in bioanalytical microdevices due to their controlled manipulation with external magnets. Current methods for creating magnetic microdevices yield a low-transparency material preventing light microscopy-based inspection of biological specimens on the structures. Uniformly transparent magnetic photoresists were developed for microdevices that require high transparency as well as consistent magnetism across the structure. Colloidal formation of 10 nm maghemite particles was minimized during addition to the negative photoresists SU-8 and 1002F through organic capping of the nanoparticles and utilization of solvent-based dispersion techniques. Photoresists with maghemite concentrations of 0.01 to 1% had a high transparency due to the even dispersal of maghemite nanoparticles within the polymer as observed with transmission electron microscopy (TEM). These magnetic photoresists were used to fabricate microstructures with aspect ratios up to 4:1 and a resolution of 3 μm. Various cell lines showed excellent adhesion and viability on the magnetic photoresists. An inspection of cells cultured on the magnetic photoresists with TEM showed cellular uptake of magnetic nanoparticles leeched from the photoresists. Cellular contamination by magnetic nanoparticles was eliminated by capping the magnetic photoresist surface with native 1002F photoresist or by removing the top layer of the magnetic photoresist through surface roughening. The utility of these magnetic photoresists was demonstrated by sorting single cells (HeLa, RBL and 3T3 cells) cultured on arrays of releasable magnetic micropallets. 100% of magnetic micropallets with attached cells were collected following release from the array. 85–92% of the collected cells expanded into colonies. The polymeric magnetic materials should find wide use in the fabrication of microstructures for bioanalytical technologies

Isolation and in Vitro Culture of Rare Cancer Stem Cells from Patient-Derived Xenografts of Pancreatic Ductal Adenocarcinoma

Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 minutes using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:104 to 1:106 on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly(dimethylsiloxane (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%) and viability (94 – 100%) for rare cell isolation. As a demonstration of the technology’s value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44 and CD326; surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44 and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90 ± 8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm2) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 μL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm2) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization

Consensus-based technical recommendations for clinical translation of renal ASL MRI

Objectives: To develop technical recommendations for the acquisition, processing and analysis of renal ASL data in the human kidney at 1.5T and 3T field strengths that can promote standardization of renal perfusion measurements and facilitate the comparability of results across scanners and in multi-center clinical studies.Methods: An international panel of 23 renal ASL experts followed a modified Delphi process, including on-line surveys and two in-person meetings, to formulate a series of consensus statements regarding patient preparation, hardware, acquisition protocol, analysis steps and data reporting.Results: Fifty-nine statements achieved consensus, while agreement could not be reached on two statements related to patient preparation. As a default protocol, the panel recommends pseudo-continuous (PCASL) or flow-sensitive alternating inversion recovery (FAIR) labeling with a single-slice spin-echo EPI readout with background suppression, and a simple but robust quantification model.Discussion: This approach is considered robust and reproducible and can provide renal perfusion images of adequate quality and SNR for most applications. If extended kidney coverage is desirable, a 2D multislice readout is recommended. These recommendations are based on current available evidence and expert opinion. Nonetheless they are expected to be updated as more data becomes available, since the renal ASL literature is rapidly expanding

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements