A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture.

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

Authentication of Coffea arabica Varieties through DNA Fingerprinting and its Significance for the Coffee Sector.

BACKGROUND: Locating the optimal varieties for coffee cultivation is increasingly considered a key condition for sustainable production and marketing. Variety performance varies when it comes to susceptibility to coffee leaf rust and other diseases, adaptation to climate change and high cup quality for specialty markets. But because of poor organization and the lack of a professional coffee seed sector, most existing coffee farms (and even seed lots and nurseries) do not know which varieties they are using. DNA fingerprinting of coffee planting material will contribute to professionalize the coffee seed sector. OBJECTIVE: The objective of this paper is i) to check in a large scale the robustness of the existing coffee DNA fingerprinting method based on eight Single Sequence Repeats markers (SRR) and ii) to describe how it can help in moving the needle towards a more professional seed sector. METHOD: 2533 samples representing all possible genetic background of Arabica varieties were DNA fingerprinted with 8 SRR markers. The genetic diversity was analyzed and the genetic conformity to varietal references was assessed. RESULTS: The DNA fingerprinting method proved to be robust in authenticating varieties and trace back the history of C. arabica breeding and of the movement of C. arabica varieties. The genetic conformity of two important coffee varieties, Marseillesa and Gesha, proved to be 91% and 39% respectively. CONCLUSIONS: DNA fingerprinting provides different actors in the coffee sector with a powerful new tool-farmers can verify the identity of their cultivated varieties, coffee roasters can be assured that marketing claims related to varieties are correct, and most of all, those looking to establish the a more professional and reliable coffee seed sector have a reliable new monitoring tool to establish and check genetic purity of seed stock and nursery plants. HIGHLIGHTS: While C. arabica is primarily self-pollinating, even fixed line varieties appear to be drifting away from their original genetic reference due to uncontrolled cross pollination. A set of 8 SSR markers applied to the largest possible genetically diverse set of samples prove to discriminate between a wide range of varieties Figures confirm that genetic non conformity of coffee varieties can represent up to 61% of checked samples.

Accurate Differentiation of Green Beans of Arabica and Robusta Coffee Using Nanofluidic Array of Single Nucleotide Polymorphism (SNP) Markers.

Green (unroasted) coffee is one of the most traded agricultural commodities in the world. The Arabica (Coffea arabica L.) and Robusta (Coffea canephora Pierre ex A. Froehner) species are the two main types of coffees for commercial production. In general, Arabica coffee is known to have better quality in terms of sensory characteristics; thus, it has a higher market value than Robusta coffee. Accurate differentiation of green beans of the two species is, therefore, of commercial interest in the coffee industry. Using the newly developed single nucleotide polymorphism (SNP) markers, we analyzed a total of 80 single green bean samples, representing 20 Arabica cultivars and four Robusta accessions. Reliable SNP fingerprints were generated for all tested samples. Unambiguous differentiation between Robusta and Arabica coffees was achieved using multivariate analysis and assignment test. The SNP marker panel and the genotyping protocol are sufficiently robust to detect admixture of green coffee in a high-throughput fashion. Moreover, the multilocus SNP approach can differentiate every single bean within Robusta and 55% of Arabica samples. This advantage, together with the single-bean sensitivity, suggests a significant potential for practical application of this technology in the coffee industry.

A single polyploidization event at the origin of the tetraploid genome of Coffea arabica is responsible for the extremely low genetic variation in wild and cultivated germplasm.

The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma's D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors.

Metal and Polymeric Strain Gauges for Si-Based, Monolithically Fabricated Organs-on-Chips.

Organ-on-chip (OOC) is becoming the alternative tool to conventional in vitro screening. Heart-on-chip devices including microstructures for mechanical and electrical stimulation have been demonstrated to be advantageous to study structural organization and maturation of heart cells. This paper presents the development of metal and polymeric strain gauges for in situ monitoring of mechanical strain in the Cytostretch platform for heart-on-chip application. Specifically, the optimization of the fabrication process of metal titanium (Ti) strain gauges and the investigation on an alternative material to improve the robustness and performance of the devices are presented. The transduction behavior and functionality of the devices are successfully proven using a custom-made set-up. The devices showed resistance changes for the pressure range (0-3 kPa) used to stretch the membranes on which heart cells can be cultured. Relative resistance changes of approximately 0.008% and 1.2% for titanium and polymeric strain gauges are respectively reported for membrane deformations up to 5%. The results demonstrate that both conventional IC metals and polymeric materials can be implemented for sensing mechanical strain using robust microfabricated organ-on-chip devices.

Advances in the Electronics for Cyclic Voltammetry: the Case of Gas Detection by Using Microfabricated Electrodes.

This paper presents an advanced voltammetric system to be used as electronic tongue for liquid and gas analysis. It has been designed to be more flexible and accurate with respect to other existing and similar systems. It features improved electronics and additional operative conditions. Among others these include the possibility to optically excite the solution and to treat the output signal by a differentiation process in order to better evidence the existence of small details in the response curve. Finally by the same type of tongue preliminary results are shown dealing with O2 and CO2 concentration measurements in appropriate solutions.

Cytostretch, an Organ-on-Chip Platform.

Organ-on-Chips (OOCs) are micro-fabricated devices which are used to culture cells in order to mimic functional units of human organs. The devices are designed to simulate the physiological environment of tissues in vivo. Cells in some types of OOCs can be stimulated in situ by electrical and/or mechanical actuators. These actuations can mimic physiological conditions in real tissue and may include fluid or air flow, or cyclic stretch and strain as they occur in the lung and heart. These conditions similarly affect cultured cells and may influence their ability to respond appropriately to physiological or pathological stimuli. To date, most focus has been on devices specifically designed to culture just one functional unit of a specific organ: lung alveoli, kidney nephrons or blood vessels, for example. In contrast, the modular Cytostretch membrane platform described here allows OOCs to be customized to different OOC applications. The platform utilizes silicon-based micro-fabrication techniques that allow low-cost, high-volume manufacturing. We describe the platform concept and its modules developed to date. Membrane variants include membranes with (i) through-membrane pores that allow biological signaling molecules to pass between two different tissue compartments; (ii) a stretchable micro-electrode array for electrical monitoring and stimulation; (iii) micro-patterning to promote cell alignment; and (iv) strain gauges to measure changes in substrate stress. This paper presents the fabrication and the proof of functionality for each module of the Cytostretch membrane. The assessment of each additional module demonstrate that a wide range of OOCs can be achieved.