Chemosensitivity of 3D Pancreatic Cancer Organoids Is Not Affected by Transformation to 2D Culture or Switch to Physiological Culture Medium.

Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10-3 ± 2.3 ×10-3 vs. 3D: 1.1 ×10-3 ± 0.6 ×10-3, p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media (9.8 × 10-3 ± 0.7 × 10-3 vs. 5.2 × 10-3 ± 1.8 × 10-3, p = 0.015). Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.

A thermophoresis-based biosensor for real-time detection of inorganic phosphate during enzymatic reactions.

Inorganic phosphate (Pi)-sensing is a key application in many disciplines, and biosensors emerged as powerful analytic tools for use in environmental Pi monitoring, food quality control, basic research, and medical diagnosis. Current sensing techniques exploit either electrochemical or optical detection approaches for Pi quantification. Here, by combining the advantages of a biological Pi-receptor based on the bacterial phosphate binding protein with the principle of thermophoresis, i.e. the diffusional motion of particles in response to a temperature gradient, we developed a continuous, sensitive, and versatile method for detecting and quantifying free Pi in the subnanomolar to micromolar range in sample volumes ≤10 µL. By recording entropy-driven changes in the directed net diffusional flux of the Pi-sensor in a temperature gradient at defined time intervals, we validate the method for analyzing steady-state enzymatic reactions associated with Pi liberation in real-time for adenosine triphosphate (ATP) turnover by myosin, the actomyosin system and for insoluble, high molecular weight enzyme-protein assemblies in biopsy derived myofibrils. Particular features of the method are: (1) high Pi-sensitivity and selectivity, (2) uncoupling of the read-out signal from potential chemical and spectroscopic interferences, (3) minimal sample volumes and nanogram protein amounts, (4) possibility to run several experiments in parallel, and (5) straightforward data analysis. The present work establishes thermophoresis as powerful sensing method in microscale format for a wide range of applications, augmenting the current set of detection principles in biosensor technology.